1 RNA SPLICING AND PROCESSING

2 hnRNA是由许多小球状结构组成的核糖核蛋白颗粒24.1  Introduction  引言 Pre-mRNA is used to describe the nuclear transcript that is processed by modification and splicing to give an mRNA. RNA splicing is the process of excising introns from RNA and connecting the exons into a continuous mRNA. Heterogeneous nuclear RNA (hnRNA) comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability. An hnRNP is the ribonucleoprotein form of hnRNA (heterogeneous nuclear RNA), in which the hnRNA is complexed with proteins. Since pre-mRNAs are not exported until processing is complete, hnRNPs are found only in the nucleus. Interrupted genes are found in all classes of organisms. They represent a minor proportion of the genes of the very lowest eukaryotes, but the vast majority of genes in higher eukaryotic genomes. Genes vary widely according to the numbers and lengths of introns, but a typical mammalian gene has 7-8 exons spread out over ~16 kb. The exons are relatively short (~ bp), and the introns are relatively long (>1 kb) (see Genes show a wide distribution of sizes). The discrepancy between the interrupted organization of the gene and the uninterrupted organization of its mRNA requires processing of the primary transcription product. The primary transcript has the same organization as the gene, and is sometimes called the pre-mRNA. Removal of the introns from pre-mRNA leaves a typical messenger of ~2.2 kb. The process by which the introns are removed is called RNA splicing. Removal of introns is a major part of the production of RNA in all eukaryotes. (Although interrupted genes are relatively rare in lower eukaryotes such as yeast, the overall proportion underestimates the importance of introns, because most of the genes that are interrupted code for relatively abundant proteins. Splicing is therefore involved in the production of a greater proportion of total mRNA than would be apparent from analysis of the genome, perhaps as much as 50%.) One of the first clues about the nature of the discrepancy in size between nuclear genes and their products in higher eukaryotes was provided by the properties of nuclear RNA. Its average size is much larger than mRNA, it is very unstable, and it has a much greater sequence complexity. Taking its name from its broad size distribution, it was called heterogeneous nuclear RNA (hnRNA). It includes pre-mRNA, but could also include other transcripts (that is, which are not ultimately processed to mRNA; for review see 17). The physical form of hnRNA is a ribonucleoprotein particle (hnRNP), in which the hnRNA is bound by proteins. As characterized in vitro, an hnRNP particle takes the form of beads connected by a fiber. The structure is summarized in Figure The most abundant proteins in the particle are the core proteins, but other proteins are present at lower stoichiometry, making a total of ~20 proteins. The proteins typically are present at ~108 copies per nucleus, compared with ~106 molecules of hnRNA. Some of the proteins may have a structural role in packaging the hnRNA; several are known to shuttle between the nucleus and cytoplasm, and play roles in exporting the RNA or otherwise controlling its activity (for review see 249; 3428). Splicing occurs in the nucleus, together with the other modifications that are made to newly synthesized RNAs. The process of expressing an interrupted gene is reviewed in Figure The transcript is capped at the 5′ end (see The 5 end of eukaryotic mRNA is capped), has the introns removed, and is polyadenylated at the 3′ end (see The 3 terminus is polyadenylated). The RNA is then transported through nuclear pores to the cytoplasm, where it is available to be translated. With regard to the various processing reactions that occur in the nucleus, we should like to know at what point splicing occurs vis-À-vis the other modifications of RNA. Does splicing occur at a particular location in the nucleus; and is it connected with other events, for example, nucleocytoplasmic transport? Does the lack of splicing make an important difference in the expression of uninterrupted genes? With regard to the splicing reaction itself, one of the main questions is how its specificity is controlled. What ensures that the ends of each intron are recognized in pairs so that the correct sequence is removed from the RNA? Are introns excised from a precursor in a particular order? Is the maturation of RNA used to regulate gene expression by discriminating among the available precursors or by changing the pattern of splicing? We can identify several types of splicing systems: Introns are removed from the nuclear pre-mRNAs of higher eukaryotes by a system that recognizes only short consensus sequences conserved at exon-intron boundaries and within the intron. This reaction requires a large splicing apparatus, which takes the form of an array of proteins and ribonucleoproteins that functions as a large particulate complex (the spliceosome). The mechanism of splicing involves transesterifications, and the catalytic center includes RNA as well as proteins. Certain RNAs have the ability to excise their introns autonomously. Introns of this type fall into two groups, as distinguished by secondary/tertiary structure. Both groups use transesterification reactions in which the RNA is the catalytic agent (see Catalytic RNA). The removal of introns from yeast nuclear tRNA precursors involves enzymatic activities that handle the substrate in a way resembling the tRNA processing enzymes, in which a critical feature is the conformation of the tRNA precursor. These splicing reactions are accomplished by enzymes that use cleavage and ligation. hnRNA是由许多小球状结构组成的核糖核蛋白颗粒

3 在细胞核中,mRNA的加工包括3和5修饰以及内含子的去除等. 剪接需要在内含子与外显子交界处产生断裂,然后将外显子末端连接在细胞核中,mRNA的加工包括3和5修饰以及内含子的去除等.剪接需要在内含子与外显子交界处产生断裂,然后将外显子末端连接.成熟的mRNA通过核孔被运到细胞质中去,在那里它完成翻译.

4 24.2 Nuclear splice junctions are short sequences. 细胞核内的RNA剪接位点是各种短序列.Splice sites are the sequences immediately surrounding the exon-intron boundaries. The GT-AG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes. Splice sites are the sequences immediately surrounding the exon-intron boundaries. They are named for their positions relative to the intron. The 5′ splice site at the 5′ (left) end of the intron includes the consensus sequence GU. The 3′ splice site at the 3′ (right) end of the intron includes the consensus sequence AG. The GU-AG rule (originally called the GT-AG rule in terms of DNA sequence) describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs. To focus on the molecular events involved in nuclear intron splicing, we must consider the nature of the splice sites, the two exon-intron boundaries that include the sites of breakage and reunion. By comparing the nucleotide sequence of mRNA with that of the structural gene, the junctions between exons and introns can be assigned. There is no extensive homology or complementarity between the two ends of an intron. However, the junctions have well conserved, though rather short, consensus sequences. It is possible to assign a specific end to every intron by relying on the conservation of exon-intron junctions. They can all be aligned to conform to the consensus sequence given in Figure The subscripts indicate the percent occurrence of the specified base at each consensus position. High conservation is found only immediately within the intron at the presumed junctions. This identifies the sequence of a generic intron as: GU……AG Because the intron defined in this way starts with the dinucleotide GU and ends with the dinucleotide AG, the junctions are often described as conforming to the GT-AG rule. (This reflects the fact that the sequences were originally analyzed in terms of DNA, but of course the GT in the coding strand sequence of DNA becomes a GU in the RNA.) Note that the two sites have different sequences and so they define the ends of the intron directionally. They are named proceeding from left to right along the intron as the 5′ splice site (sometimes called the left or donor site) and the 3′ splice site (also called the right or acceptor site). The consensus sequences are implicated as the sites recognized in splicing by point mutations that prevent splicing in vivo and in vitro (for review see 242; 243).  剪接位点是指外显子内含子交界处序列, 它们一般根据与内含子的相对位置命名. 内含子5端的5剪接位点含共有序列GU. 内含子3端的3剪接位点含共有序列AG. GU-AG规则(最初在DNA序列中被称作GT-AG规则)描述了在前体mRNA中内含子的最初及最末位置上必须出现的恒定的双碱基.

5 内含子的末端按GU-AG规则定义.

6 24.4 pre-mRNA splicing proceeds through a lariat. 前体mRNA剪接要通过套索结构.The lariat is an intermediate in RNA splicing in which a circular structure with a tail is created by a 5′-2′ bond. The branch site is a short sequence just before the end of an intron at which the lariat intermediate is formed in splicing by joining the 5′ nucleotide of the intron to the 2′ position of an Adenosine. A transesterification reaction breaks and makes chemical bonds in a coordinated transfer so that no energy is required. Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site. The branch sequence is conserved in yeast but less well conserved in higher eukaryotes. A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron. The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together. The reactions occur by transesterifications in which a bond is transferred from one location to another. The mechanism of splicing has been characterized in vitro, using systems in which introns can be removed from RNA precursors. Nuclear extracts can splice purified RNA precursors, which shows that the action of splicing is not linked to the process of transcription. Splicing can occur to RNAs that are neither capped nor polyadenylated. However, although the splicing reaction as such is independent of transcription or modification to the RNA, these events normally occur in a coordinated manner, and the efficiency of splicing may be influenced by other processing events. The stages of splicing in vitro are illustrated in the pathway of Figure We discuss the reaction in terms of the individual RNA species that can be identified, but remember that in vivo the species containing exons are not released as free molecules, but remain held together by the splicing apparatus (for review see 253). The first step is to make a cut at the 5′ splice site, separating the left exon and the right intron-exon molecule. The left exon takes the form of a linear molecule. The right intron-exon molecule forms a lariat, in which the 5′ terminus generated at the end of the intron becomes linked by a 5′ – 2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site (712). Cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon. The cleavage and ligation reactions are shown separately in the figure for illustrative purposes, but actually occur as one coordinated transfer. The lariat is then "debranched" to give a linear excised intron, which is rapidly degraded. The sequences needed for splicing are the short consensus sequences at the 5′and 3′splice sites and at the branch site. Together with the knowledge that most of the sequence of an intron can be deleted without impeding splicing, this indicates that there is no demand for specific conformation in the intron (or exon). The branch site plays an important role in identifying the 3′ splice site. The branch site in yeast is highly conserved, and has the consensus sequence UACUAAC. The branch site in higher eukaryotes is not well conserved, but has a preference for purines or pyrimidines at each position and retains the target A nucleotide (see Figure 24.6) (717). The branch site lies nucleotides upstream of the 3′ splice site. Mutations or deletions of the branch site in yeast prevent splicing. In higher eukaryotes, the relaxed constraints in its sequence result in the ability to use related sequences (called cryptic sites) when the authentic branch is deleted. Proximity to the 3′ splice site appears to be important, since the cryptic site is always close to the authentic site. A cryptic site is used only when the branch site has been inactivated. When a cryptic branch sequence is used in this manner, splicing otherwise appears to be normal; and the exons give the same products as wild type. The role of the branch site therefore is to identify the nearest 3′splice site as the target for connection to the 5′ splice site (713). This can be explained by the fact that an interaction occurs between protein complexes that bind to these two sites. The bond that forms the lariat goes from the 5′ position of the invariant G that was at the 5′ end of the intron to the 2′ position of the invariant A in the branch site. This corresponds to the third A residue in the yeast UACUAAC box. The chemical reactions proceed by transesterification: a bond is in effect transferred from one location to another. Figure 24.7 shows that the first step is a nucleophilic attack by the 2′ – OH of the invariant A of the UACUAAC sequence on the 5′ splice site. In the second step, the free 3′ – OH of the exon that was released by the first reaction now attacks the bond at the 3′ splice site. Note that the number of phosphodiester bonds is conserved. There were originally two 5′ – 3′ bonds at the exon-intron splice sites; one has been replaced by the 5′ – 3′ bond between the exons, and the other has been replaced by the 5′ – 2′ bond that forms the lariat (for review see 251).  当5剪接位点被剪开,且内含子5端连接到内含子分支位点上的A的2位置时,将形成套索结构. 当3剪接位点被剪开,内含子将以套索的形式被释放,其左右的外显子将会连接在一起.

7 5和3剪接位点以及分支位点对剪接过程而言是充分必要条件. 分支位点的序列在酵母中是完全保守的,但在较高等的真核生物中,其保守性则不是很高. The lariat is an intermediate in RNA splicing in which a circular structure with a tail is created by a 5′-2′ bond. The branch site is a short sequence just before the end of an intron at which the lariat intermediate is formed in splicing by joining the 5′ nucleotide of the intron to the 2′ position of an Adenosine. A transesterification reaction breaks and makes chemical bonds in a coordinated transfer so that no energy is required. Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site. The branch sequence is conserved in yeast but less well conserved in higher eukaryotes. A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron. The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together. The reactions occur by transesterifications in which a bond is transferred from one location to another. The mechanism of splicing has been characterized in vitro, using systems in which introns can be removed from RNA precursors. Nuclear extracts can splice purified RNA precursors, which shows that the action of splicing is not linked to the process of transcription. Splicing can occur to RNAs that are neither capped nor polyadenylated. However, although the splicing reaction as such is independent of transcription or modification to the RNA, these events normally occur in a coordinated manner, and the efficiency of splicing may be influenced by other processing events. The stages of splicing in vitro are illustrated in the pathway of Figure We discuss the reaction in terms of the individual RNA species that can be identified, but remember that in vivo the species containing exons are not released as free molecules, but remain held together by the splicing apparatus (for review see 253). The first step is to make a cut at the 5′ splice site, separating the left exon and the right intron-exon molecule. The left exon takes the form of a linear molecule. The right intron-exon molecule forms a lariat, in which the 5′ terminus generated at the end of the intron becomes linked by a 5′ – 2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site (712). Cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon. The cleavage and ligation reactions are shown separately in the figure for illustrative purposes, but actually occur as one coordinated transfer. The lariat is then "debranched" to give a linear excised intron, which is rapidly degraded. The sequences needed for splicing are the short consensus sequences at the 5′and 3′splice sites and at the branch site. Together with the knowledge that most of the sequence of an intron can be deleted without impeding splicing, this indicates that there is no demand for specific conformation in the intron (or exon). The branch site plays an important role in identifying the 3′ splice site. The branch site in yeast is highly conserved, and has the consensus sequence UACUAAC. The branch site in higher eukaryotes is not well conserved, but has a preference for purines or pyrimidines at each position and retains the target A nucleotide (see Figure 24.6) (717). The branch site lies nucleotides upstream of the 3′ splice site. Mutations or deletions of the branch site in yeast prevent splicing. In higher eukaryotes, the relaxed constraints in its sequence result in the ability to use related sequences (called cryptic sites) when the authentic branch is deleted. Proximity to the 3′ splice site appears to be important, since the cryptic site is always close to the authentic site. A cryptic site is used only when the branch site has been inactivated. When a cryptic branch sequence is used in this manner, splicing otherwise appears to be normal; and the exons give the same products as wild type. The role of the branch site therefore is to identify the nearest 3′splice site as the target for connection to the 5′ splice site (713). This can be explained by the fact that an interaction occurs between protein complexes that bind to these two sites. The bond that forms the lariat goes from the 5′ position of the invariant G that was at the 5′ end of the intron to the 2′ position of the invariant A in the branch site. This corresponds to the third A residue in the yeast UACUAAC box. The chemical reactions proceed by transesterification: a bond is in effect transferred from one location to another. Figure 24.7 shows that the first step is a nucleophilic attack by the 2′ – OH of the invariant A of the UACUAAC sequence on the 5′ splice site. In the second step, the free 3′ – OH of the exon that was released by the first reaction now attacks the bond at the 3′ splice site. Note that the number of phosphodiester bonds is conserved. There were originally two 5′ – 3′ bonds at the exon-intron splice sites; one has been replaced by the 5′ – 3′ bond between the exons, and the other has been replaced by the 5′ – 2′ bond that forms the lariat (for review see 251). 

8 剪接反应分两个阶段,5外显子首先被切开, 然后与另一个外显子的5相连.

9 24.5 snRNAs are required for splicing. snRNA是剪接必需的.参与剪接过程的5个snRNP是U1, U2, U4, U5, 和U6. snRNP和其他一些辅助蛋白质共同构成了剪接体. A small nuclear RNA (snRNA) is one of many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions. Small cytoplasmic RNAs (scRNA) are present in the cytoplasm and (sometimes are also found in the nucleus). snRNPs (snurp) are small nuclear ribonucleoproteins (snRNAs associated with proteins). scRNPs (scyrp) are small cytoplasmic ribonucleoproteins (scRNAs associated with proteins). The spliceosome is a complex formed by the snRNPs that are required for splicing together with additional protein factors. Anti-Sm is an autoimmune antiserum that defines the Sm epitope that is common to a group of proteins found in snRNPs that are involved in RNA splicing. The five snRNPs involved in splicing are U1, U2, U5, U4, and U6. Together with some additional proteins, the snRNPs form the spliceosome. All the snRNPs except U6 contain a conserved sequence that binds the Sm proteins that are recognized by antibodies generated in autoimmune disease. The 5′ and 3′ splice sites and the branch sequence are recognized by components of the splicing apparatus that assemble to form a large complex. This complex brings together the 5′ and 3′ splice sites before any reaction occurs, explaining why a deficiency in any one of the sites may prevent the reaction from initiating. The complex assembles sequentially on the pre-mRNA, and several intermediates can be recognized by fractionating complexes of different sizes. Splicing occurs only after all the components have assembled (719). The splicing apparatus contains both proteins and RNAs (in addition to the pre-mRNA). The RNAs take the form of small molecules that exist as ribonucleoprotein particles. Both the nucleus and cytoplasm of eukaryotic cells contain many discrete small RNA species. They range in size from bases in higher eukaryotes, and extend in length to ~1000 bases in yeast. They vary considerably in abundance, from molecules per cell to concentrations too low to be detected directly. Those restricted to the nucleus are called small nuclear RNAs (snRNA); those found in the cytoplasm are called small cytoplasmic RNAs (scRNA). In their natural state, they exist as ribonucleoprotein particles (snRNP and scRNP). Colloquially, they are sometimes known as snurps and scyrps. There is also a class of small RNAs found in the nucleolus, called snoRNAs, which are involved in processing ribosomal RNA (see Small RNAs are required for rRNA processing). The snRNPs involved in splicing, together with many additional proteins, form a large particulate complex, called the spliceosome. Isolated from the in vitro splicing systems, it comprises a 50-60S ribonucleoprotein particle. The spliceosome may be formed in stages as the snRNPs join, proceeding through several "presplicing complexes." The spliceosome is a large body, greater in mass than the ribosome. Figure 24.8 summarizes the components of the spliceosome (3210). The 5 snRNAs account for more than a quarter of the mass; together with their 41 associated proteins, they account for almost half of the mass. Some 70 other proteins found in the spliceosome are described as splicing factors. They include proteins required for assembly of the spliceosome, proteins required for it to bind to the RNA substrate, and proteins involved in the catalytic process. In addition to these proteins, another ~30 proteins associated with the spliceosome have been implicated in acting at other stages of gene expression, suggesting that the spliceosome may serve as a coordinating apparatus. The spliceosome forms on the intact precursor RNA and passes through an intermediate state in which it contains the individual 5′ exon linear molecule and the right lariat-intron-exon. Little spliced product is found in the complex, which suggests that it is usually released immediately following the cleavage of the 3′ site and ligation of the exons. We may think of the snRNP particles as being involved in building the structure of the spliceosome. Like the ribosome, the spliceosome depends on RNA-RNA interactions as well as protein-RNA and protein-protein interactions. Some of the reactions involving the snRNPs require their RNAs to base pair directly with sequences in the RNA being spliced; other reactions require recognition between snRNPs or between their proteins and other components of the spliceosome. The importance of snRNA molecules can be tested directly in yeast by making mutations in their genes. Mutations in 5 snRNA genes are lethal and prevent splicing. All of the snRNAs involved in splicing can be recognized in conserved forms in animal, bird, and insect cells. The corresponding RNAs in yeast are often rather larger, but conserved regions include features that are similar to the snRNAs of higher eukaryotes. The snRNPs involved in splicing are U1, U2, U5, U4, and U6. They are named according to the snRNAs that are present. Each snRNP contains a single snRNA and several (<20) proteins. The U4 and U6 snRNPs are usually found as a single (U4/U6) particle. A common structural core for each snRNP consists of a group of 8 proteins, all of which are recognized by an autoimmune antiserum called anti-Sm; conserved sequences in the proteins form the target for the antibodies. The other proteins in each snRNP are unique to it. The Sm proteins bind to the conserved sequence PuAU36Gpu, which is present in all snRNAs except U6. The U6 snRNP contains instead a set of Sm-like (Lsm) proteins. The Sm proteins must be involved in the autoimmune reaction, although their relationship to the phenotype of the autoimmune disease is not clear (for review see 244; 245; 247). Some of the proteins in the snRNPs may be involved directly in splicing; others may be required in structural roles or just for assembly or interactions between the snRNP particles. About one third of the proteins involved in splicing are components of the snRNPs. Increasing evidence for a direct role of RNA in the splicing reaction suggests that relatively few of the splicing factors play a direct role in catalysis; most are involved in structural or assembly roles.

10 24.6 U1 snRNP initiates splicing. U1 snRNP启动剪接.U1 snRNP通过RNA-RNA配对反应与5剪接位点结合, 从而起始剪接过程. E复合体包括结合在5剪接位点的U1 snRNP, 结合在分支位点与3剪接位点间嘧啶富集区的U2AF和连接U1 snRNP与U2AF的SR蛋白. An SR protein has a variable length of an Arg-Ser-rich region and is involved in splicing. U1 snRNP initiates splicing by binding to the 5′ splice site by means of an RNA-RNA pairing reaction. The E complex contains U1 snRNP bound at the 5′ splice site, the protein U2AF bound to a pyrimidine tract between the branch site and the 3′ splice site, and SR proteins connecting U1 snRNP to U2AF. Splicing can be broadly divided into two stages: First the consensus sequences at the 5′ splice site, branch sequence, and adjacent pyrimidine tract are recognized. A complex assembles that contains all of the splicing components. Then the cleavage and ligation reactions change the structure of the substrate RNA. Components of the complex are released or reorganized as it proceeds through the splicing reactions. The important point is that all of the splicing components are assembled and have assured that the splice sites are available before any irreversible change is made to the RNA (for review see 3239). Recognition of the consensus sequences involves both RNAs and proteins. Certain snRNAs have sequences that are complementary to the consensus sequences or to one another, and base pairing between snRNA and pre-mRNA, or between snRNAs, plays an important role in splicing. The human U1 snRNP contains 8 proteins as well as the RNA. The secondary structure of the U1 snRNA is drawn in Figure It contains several domains. The Sm-binding site is required for interaction with the common snRNP proteins. Domains identified by the individual stem-loop structures provide binding sites for proteins that are unique to U1 snRNP. Binding of U1 snRNP to the 5′ splice site is the first step in splicing. The recruitment of U1 snRNP involves an interaction between one of its proteins (U1-70k) and the protein ASF/SF2 (a general splicing factor in the SR class: see below). U1 snRNA base pairs with the 5′ site by means of a single-stranded region at its 5′ – terminus which usually includes a stretch of 4-6 bases that is complementary with the splice site. Mutations in the 5′ splice site and U1 snRNA can be used to test directly whether pairing between them is necessary. The results of such an experiment are illustrated in Figure The wild-type sequence of the splice site of the 12S adenovirus pre-mRNA pairs at 5 out of 6 positions with U1 snRNA. A mutant in the 12S RNA that cannot be spliced has two sequence changes; the GG residues at positions 5-6 in the intron are changed to AU. The mutation changes the pattern of base pairing between U1 snRNA and the 5′ splice site, although it does not alter the overall extent of pairing (because complementarity is lost at one position and gained at the other). The effect on splicing suggests that the base-pairing interaction is important. When a mutation is introduced into U1 snRNA that restores pairing at position 5, normal splicing is regained. Other cases in which corresponding mutations are made in U1 snRNA to see whether they can suppress the mutation in the splice site suggests the general rule: complementarity between U1 snRNA and the 5′ splice site is necessary for splicing, but the efficiency of splicing is not determined solely by the number of base pairs that can form (714). The pairing reaction is stabilized by the proteins of the U1 snRNP (3246). Figure shows the early stages of splicing. The first complex formed during splicing is the E (early presplicing) complex, which contains U1 snRNP, the splicing factor U2AF, and members of a family called SR proteins, which comprise an important group of splicing factors and regulators. They take their name from the presence of an Arg-Ser-rich region that is variable in length. SR proteins interact with one another via their Arg-Ser-rich regions. They also bind to RNA. They are an essential component of the spliceosome, forming a framework on the RNA substrate. They connect U2AF to U1 (see Figure 24.12). The E complex is sometimes called the commitment complex, because its formation identifies a pre-mRNA as a substrate for formation of the splicing complex. In the E complex, U2AF is bound to the region between the branch site and the 3′ splice site. The name of U2AF reflects its original isolation as the U2 auxiliary factor. In most organisms, it has a large subunit (U2AF65) that contacts a pyrimidine tract downstream of the branch site, while a small subunit (U2AF35) directly contacts the dinucleotide AG at the 3′ splice site (3247; 3248; 3249). In S. cerevisiae, this function is filled by the protein Mud2, which is a counterpart of U2AF65, and binds only to the pyrimidine tract. This marks a difference in the mechanism of splicing between S. cerevisiae and other organisms. In the yeast, the 3′ splice site is not involved in the early stages of forming the splicing complex, but in all other known cases, it is required. Another splicing factor, called SF1 in mammals and BBP in yeast, connects U2AF/Mud2 to the U1 snRNP bound at the 5′ splice site (3250; 3251). Complex formation is enhanced by the cooperative reactions of the two proteins; SF1 and U2AF (or BBP and Mud2) bind together to the RNA substrate ~10× more effectively than either alone. This interaction is probably responsible for making the first connection between the two splice sites across the intron. The E complex is converted to the A complex when U2 snRNP binds to the branch site. Both U1 snRNP and U2AF/Mud2 are needed for U2 binding. The U2 snRNA includes sequences complementary to the branch site. A sequence near the 5′ end of the snRNA base pairs with the branch sequence in the intron. In yeast this typically involves formation of a duplex with the UACUAAC box (see Figure 24.14). Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site. The addition of U2 snRNP to the E complex generates the A presplicing complex. The binding of U2 snRNP requires ATP hydrolysis, and commits a pre-mRNA to the splicing pathway (1560; 718).

11 人类U1 snRNP有一个可产生数个结构域的配对结构, 其5端保留着单链形式, 可以与5剪接位点配对.

12 E复合物由三种物质连续地加入而成, 即U1-snRNP加到5剪接位点, U2AF加到嘧啶区/3剪接位点, 以及桥连蛋白SF1/BBP的加入.

13 形成E复合体的直接方法是U1 snRNP结合在5剪接位点且U2AF结合在分支位点和3剪接位点之间的嘧啶区.24.7 The E complex can be formed by intron definition or exon definition. E复合物可通过内含子定界或外显子定界来形成. 形成E复合体的直接方法是U1 snRNP结合在5剪接位点且U2AF结合在分支位点和3剪接位点之间的嘧啶区. 另一个可能是在嘧啶区的U2AF和下游5剪接位点的U1-snRNP之间形成复合体. 当U2 snRNP结合在分支位点时, E复合体转变成A复合体. Intron definition describes the process when a pair of splicing sites are recognized by interactions involving only the 5′ site and the branchpoint/3′ site. Exon definition describes the process when a pair of splicing sites are recognized by interactions involving the 5′ site of the intron and also the 5′ site of the next intron downstream. The direct way of forming an E complex is for U1 snRNP to bind at the 5′ splice site and U2AF to bind at a pyrimidine tract between the branch site and the 3′ splice site. Another possibility is for the complex to form between U2AF at the pyrimidine tract and U1 snRNP at a downstream 5′ splice site. The E complex is converted to the A complex when U2 snRNP binds at the branch site. If an E complex forms using a downstream 5′ splice site, this splice site is replaced by the appropriate upstream 5′ splice site when the E complex is converted to the A complex. Weak 3′ splice sites may require a splicing enhancer located in the exon downstream to bind SR proteins directly. There is more than one way to form the E complex. Figure illustrates some possibilities. The most direct reaction is for both splice sites to be recognized across the intron. The presence of U1 snRNP at the 5′ splice site is necessary for U2AF to bind at the pyrimidine tract downstream of the branch site, making it possible that the 5′ and 3′ ends of the intron are brought together in this complex. The E complex is converted to the A complex when U2 snRNP binds at the branch site. The basic feature of this route for splicing is that the two splice sites are recognized without requiring any sequences outside of the intron. This process is called intron definition. In an extreme case, the SR proteins may enable U2AF/U2 snRNP to bind in vitro in the absence of U1, raising the possibility that there could be a U1-independent pathway for splicing. An alternative route to form the spliceosome may be followed when the introns are long and the splice sites are weak. As shown on the right of the figure, the 5′ splice site is recognized by U1 snRNA in the usual way. However, the 3′ splice site is recognized as part of a complex that forms across the next exon, in which the next 5′ splice site is also bound by U1 snRNA. This U1 snRNA is connected by SR proteins to the U2AF at the pyrimidine tract. When U2 snRNP joins to generate the A complex, there is a rearrangement, in which the correct (leftmost) 5′ splice site displaces the downstream 5′ splice site in the complex. The important feature of this route for splicing is that sequences downstream of the intron itself are required. Usually these sequences include the next 5′ splice site. This process is called exon definition. This mechanism is not universal: neither SR proteins nor exon definition are found in S. cerevisiae. "Weak" 3′ splice sites do not bind U2AF and U2 snRNP effectively. Additional sequences are needed to bind the SR proteins, which assist U2AF in binding to the pyrimidine tract. Such sequences are called "splicing enhancers," and they are most commonly found in the exon downstream of the 3′ splice site (734). 

14 多条途径可以启动对5和3剪接位点的识别.

15 24.8 5 snRNPs form the spliceosome. 五种snRNP形成了剪接体.Binding of U5 and U4/U6 snRNPs converts the A complex to the B1 spliceosome, which contains all the components necessary for splicing. The spliceosome passes through a series of further complexes as splicing proceeds. Release of U1 snRNP allows U6 snRNA to interact with the 5′ splice site, and converts the B1 spliceosome to the B2 spliceosome. When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site. Following formation of the E complex, the other snRNPs and factors involved in splicing associate with the complex in a defined order. Figure shows the components of the complexes that can be identified as the reaction proceeds. The B1 complex is formed when a trimer containing the U5 and U4/U6 snRNPs binds to the A complex containing U1 and U2 snRNPs. This complex is regarded as a spliceosome, since it contains the components needed for the splicing reaction. It is converted to the B2 complex after U1 is released. The dissociation of U1 is necessary to allow other components to come into juxtaposition with the 5′ splice site, most notably U6 snRNA. At this point U5 snRNA changes its position; initially it is close to exon sequences at the 5′ splice site, but it shifts to the vicinity of the intron sequences (722). The catalytic reaction is triggered by the release of U4; this requires hydrolysis of ATP. The role of U4 snRNA may be to sequester U6 snRNA until it is needed. Figure shows the changes that occur in the base pairing interactions between snRNAs during splicing. In the U6/U4 snRNP, a continuous length of 26 bases of U6 is paired with two separated regions of U4. When U4 dissociates, the region in U6 that is released becomes free to take up another structure. The first part of it pairs with U2; the second part forms an intramolecular hairpin. The interaction between U4 and U6 is mutually incompatible with the interaction between U2 and U6, so the release of U4 controls the ability of the spliceosome to proceed (721; for review see 252). Although for clarity the figure shows the RNA substrate in extended form, the 5′ splice site is actually close to the U6 sequence immediately on the 5′ side of the stretch bound to U2. This sequence in U6 snRNA pairs with sequences in the intron just downstream of the conserved GU at the 5′ splice site (mutations that enhance such pairing improve the efficiency of splicing). So several pairing reactions between snRNAs and the substrate RNA occur in the course of splicing. They are summarized in Figure The snRNPs have sequences that pair with the substrate and with one another. They also have single-stranded regions in loops that are in close proximity to sequences in the substrate, and which play an important role, as judged by the ability of mutations in the loops to block splicing. The base pairing between U2 and the branch point, and between U2 and U6, creates a structure that resembles the active center of group II self-splicing introns (see Figure 24.20). This suggests the possibility that the catalytic component could comprise an RNA structure generated by the U2-U6 interaction. U6 is paired with the 5′ splice site, and crosslinking experiments show that a loop in U5 snRNA is immediately adjacent to the first base positions in both exons. But although we can define the proximities of the substrate (5′ splice site and branch site) and snurps (U2 and U6) at the catalytic center (as shown in Figure 24.14), the components that undertake the transesterifications have not been directly identified (723; 724; 725). The formation of the lariat at the branch site is responsible for determining the use of the 3′ splice site, since the 3′ consensus sequence nearest to the 3′ side of the branch becomes the target for the second transesterification. The second splicing reaction follows rapidly. Binding of U5 snRNP to the 3′ splice site is needed for this reaction, but there is no evidence for a base pairing reaction. The important conclusion suggested by these results is that the snRNA components of the splicing apparatus interact both among themselves and with the substrate RNA by means of base pairing interactions, and these interactions allow for changes in structure that may bring reacting groups into apposition and may even create catalytic centers. Furthermore, the conformational changes in the snRNAs are reversible; for example, U6 snRNA is not used up in a splicing reaction, and at completion must be released from U2, so that it can reform the duplex structure with U4 to undertake another cycle of splicing. We have described individual reactions in which each snRNP participates, but as might be expected from a complex series of reactions, any particular snRNP may play more than one role in splicing. So the ability of U1 snRNP to promote binding of U2 snRNP to the branch site is independent of its ability to bind to the 5′ splice site. Similarly, different regions of U2 snRNA can be defined that are needed to bind to the branch site and to interact with other splicing components. An extensive mutational analysis has been undertaken in yeast to identify both the RNA and protein components of the spliceosome. Mutations in genes needed for splicing are identified by the accumulation of unspliced precursors. A series of loci that identify genes potentially coding for proteins involved in splicing were originally called RNA, but are now known as PRP mutants (for pre-RNA processing). Several of the products of these genes have motifs that identify them as RNA-binding proteins, and some appear to be related to a family of ATP-dependent RNA helicases. We suppose that, in addition to RNA-RNA interactions, protein-RNA interactions are important in creating or releasing structures in the pre-mRNA or snRNA components of the spliceosomes. Some of the PRP proteins are components of snRNP particles, but others function as independent factors. One interesting example is PRP16, a helicase that hydrolyzes ATP, and associates transiently with the spliceosome to participate in the second catalytic step. Another example is PRP22, another ATP-dependent helicase, which is required to release the mature mRNA from the spliceosome. The conservation of bonds during the splicing reaction means that input of energy is not required to drive bond formation per se, which implies that the ATP hydrolysis is required for other purposes. The use of ATP by PRP16 and PRP22 may be examples of a more general phenomenon: the use of ATP hydrolysis to drive conformational changes that are needed to proceed through splicing (for review see 254). U5和U4/U6 snRNP的结合把A复合体变成B1剪接体, 其中包含了所有剪接所必需的元件. 剪接体在剪接过程中经历了一系列其他的复合体形式.

16 剪接反应经过几个互相独立的阶段, 在此过程中, 能够识别共有序列的各组分之间的相互作用导致剪接体的形成.

17 24.10 Splicing is connected to export of mRNA. 剪接与mRNA出核相关联.REF蛋白通过和剪接体的连接而结合到剪接位点. 剪接后, 它们依旧附着于RNA外显子-外显子连接处. 它们和转运蛋白TAP/Mex相互作用, 并将RNA运出核孔. The REF proteins bind to splicing junctions by associating with the spliceosome. After splicing, they remain attached to the RNA at the exon-exon junction. They interact with the transport protein TAP/Mex that exports the RNA through the nuclear pore. After it has been synthesized and processed, mRNA is exported from the nucleus to the cytoplasm in the form of a ribonucleoprotein complex. The proteins that are responsible for transport "shuttle" between the nucleus and cytoplasm, remaining in the compartment only briefly (see Transport receptors carry cargo proteins through the pore). One important question is how these proteins recognize their RNA substrates, and what ensures that only fully processed mRNAs are exported. Part of the answer may lie in the relative timing of events: spliceosomes may form to remove introns before transcription has been completed. However, there may also be a direct connection between splicing and export. Introns may prevent export of mRNA because they are associated with the splicing apparatus. The spliceosome also may provide the initial point of contact for the export apparatus. Figure shows a model in which a protein complex binds to the RNA via the splicing apparatus. The complex consists of >9 proteins and is called the EJC (exon junction complex). The EJC is involved in several functions of spliced mRNAs (for review see 3428). Some of the proteins of the EJC are directly involved in these functions, and others recruit additional proteins for particular functions. The first contact in assembling the EJC is made with one of the splicing factors (2072; 2073; 2074). Then after splicing, the EJC remains attached to the mRNA just upstream of the exon-exon junction (2071; 2075; 3232; 3233). The EJC is not associated with RNAs transcribed from genes that lack introns, so its involvement in the process is unique for spliced products. If introns are deleted from a gene, its RNA product is exported much more slowly to the cytoplasm (2069). This suggests that the intron may provide a signal for attachment of the export apparatus. We can now account for this phenomenon in terms of a series of protein interactions, as shown in Figure The EJC includes a group of proteins called the REF family (the best characterized member is called Aly) (2070). The REF proteins in turn interact with a transport protein (variously called TAP and Mex) which has direct responsibility for interaction with the nuclear pore (for review see 2422). A similar system may be used to identify a spliced RNA so that nonsense mutations prior to the last exon trigger its degradation in the cytoplasm (see Nonsense mutations trigger a surveillance system). 

18 外显子连接复合体通过识别剪接复合体结合到RNA上.

19 REF蛋白结合剪接因子并保留在剪接产物上. REF结合到位于核孔的出核因子上.

20 II类内含子通过自我催化剪接活动从RNA上切除自身. II类内含子的剪接点和剪接机制同细胞核内含子相似.24.11 Group II introns autosplice via lariat formation.  II类内含子通过套索结构进行自我剪接. II类内含子通过自我催化剪接活动从RNA上切除自身. II类内含子的剪接点和剪接机制同细胞核内含子相似. II类内含子折叠成二级结构以产生催化位点, 这个位点类似于U6-U2-细胞核内含子. Autosplicing (Self-splicing) describes the ability of an intron to excise itself from an RNA by a catalytic action that depends only on the sequence of RNA in the intron. Group II introns excise themselves from RNA by an autocatalytic splicing event. The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns. A group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron. Introns in protein-coding genes (in fact, in all genes except nuclear tRNA-coding genes) can be divided into three general classes. Nuclear pre-mRNA introns are identified only by the possession of the GU...AG dinucleotides at the 5′ and 3′ ends and the branch site/pyrimidine tract near the 3′ end. They do not show any common features of secondary structure. Group I and group II introns are found in organelles and in bacteria. (Group I introns are found also in the nucleus in lower eukaryotes.) Group I and group II introns are classified according to their internal organization. Each can be folded into a typical type of secondary structure. The group I and group II introns have the remarkable ability to excise themselves from an RNA. This is called autosplicing. Group I introns are more common than group II introns. There is little relationship between the two classes, but in each case the RNA can perform the splicing reaction in vitro by itself, without requiring enzymatic activities provided by proteins; however, proteins are almost certainly required in vivo to assist with folding (see Catalytic RNA). Figure shows that three classes of introns are excised by two successive transesterifications (shown previously for nuclear introns in Figure 24.6). In the first reaction, the 5′ exon-intron junction is attacked by a free hydroxyl group (provided by an internal 2′ – OH position in nuclear and group II introns, and by a free guanine nucleotide in group I introns). In the second reaction, the free 3′ – OH at the end of the released exon in turn attacks the 3′ intron-exon junction. There are parallels between group II introns and pre-mRNA splicing. Group II mitochondrial introns are excised by the same mechanism as nuclear pre-mRNAs, via a lariat that is held together by a 5′ – 2′ bond. An example of a lariat produced by splicing a group II intron is shown in Figure When an isolated group II RNA is incubated in vitro in the absence of additional components, it is able to perform the splicing reaction. This means that the two transesterification reactions shown in Figure can be performed by the group II intron RNA sequence itself. Because the number of phosphodiester bonds is conserved in the reaction, an external supply of energy is not required; this could have been an important feature in the evolution of splicing (for review see 260). A group II intron forms into a secondary structure that contains several domains formed by base-paired stems and single-stranded loops. Domain 5 is separated by 2 bases from domain 6, which contains an A residue that donates the 2′ – OH group for the first transesterification. This constitutes a catalytic domain in the RNA. Figure compares this secondary structure with the structure formed by the combination of U6 with U2 and of U2 with the branch site. The similarity suggests that U6 may have a catalytic role. The features of group II splicing suggest that splicing evolved from an autocatalytic reaction undertaken by an individual RNA molecule, in which it accomplished a controlled deletion of an internal sequence. Probably such a reaction requires the RNA to fold into a specific conformation, or series of conformations, and would occur exclusively in cis conformation. The ability of group II introns to remove themselves by an autocatalytic splicing event stands in great contrast to the requirement of nuclear introns for a complex splicing apparatus. We may regard the snRNAs of the spliceosome as compensating for the lack of sequence information in the intron, and providing the information required to form particular structures in RNA. The functions of the snRNAs may have evolved from the original autocatalytic system. These snRNAs act in trans upon the substrate pre-mRNA; we might imagine that the ability of U1 to pair with the 5′ splice site, or of U2 to pair with the branch sequence, replaced a similar reaction that required the relevant sequence to be carried by the intron. So the snRNAs may undergo reactions with the pre-mRNA substrate and with one another that have substituted for the series of conformational changes that occur in RNAs that splice by group II mechanisms. In effect, these changes have relieved the substrate pre-mRNA of the obligation to carry the sequences needed to sponsor the reaction. As the splicing apparatus has become more complex (and as the number of potential substrates has increased), proteins have played a more important role.

21 三类剪接反应按两步酯交换作用进行: 首先游离羟基进攻外显子1和内含子结合处, 接着外显子1末端产生的羟基进攻外显子2与内含子结合处.

22 某些外显子可能会由于某一对剪接位点的使用与否而被包含或被剔除于RNA产物中.24.12 Alternative splicing involves differential use of splice junctions. 可变剪接使用不同的剪接位点. Alternative splicing describes the production of different RNA products from a single product by changes in the usage of splicing junctions. Specific exons may be excluded or included in the RNA product by using or failing to use a pair of splicing junctions. Exons may be extended by changing one of the splice junctions to use an alternative junction. Sex determination in Drosophila involves a series of alternative splicing events in genes coding for successive products of a pathway. P elements of Drosophila show germline-specific alternative splicing. When an interrupted gene is transcribed into an RNA that gives rise to a single type of spliced mRNA, there is no ambiguity in assignment of exons and introns. But the RNAs of some genes follow patterns of alternative splicing, when a single gene gives rise to more than one mRNA sequence. In some cases, the ultimate pattern of expression is dictated by the primary transcript, because the use of different startpoints or the generation of alternative 3′ ends alters the pattern of splicing. In other cases, a single primary transcript is spliced in more than one way, and internal exons are substituted, added, or deleted. In some cases, the multiple products all are made in the same cell, but in others the process is regulated so that particular splicing patterns occur only under particular conditions (for review see 246). One of the most pressing questions in splicing is to determine what controls the use of such alternative pathways. Proteins that intervene to bias the use of alternative splice sites have been identified in two ways. In some mammalian systems, it has been possible to characterize alternative splicing in vitro, and to identify proteins that are required for the process. In D. melanogaster, aberrations in alternative splicing may be caused either by mutations in the genes that are alternatively spliced or in the genes whose products are necessary for the reaction. Figure shows examples of alternative splicing in which one splice site remains constant, but the other varies. The large T/ small t antigens of SV40 and the products of the adenovirus E1A region are generated by connecting a varying 5′ site to a constant 3′ site. In the case of the T/t antigens, the 5′ site used for T antigen removes a termination codon that is present in the t antigen mRNA, so that T antigen is larger than t antigen. In the case of the E1A transcripts, one of the 5′ sites connects to the last exon in a different reading frame, again making a significant change in the C-terminal part of the protein. In these examples, all the relevant splicing events take place in every cell in which the gene is expressed, so all the protein products are made. There are differences in the ratios of T/t antigens in different cell types. A protein extracted from cells that produce relatively more small t antigen can cause preferential production of small t RNA in extracts from other cell types. This protein, which was called ASF (alternative splicing factor), turns out to be the same as the splicing factor SF2, which is required for early steps in spliceosome assembly and for the first cleavage-ligation reaction (see Figure 24.13). ASF/SF2 is an RNA-binding protein in the SR family. When a pre-mRNA has more than one 5′ splice site preceding a single 3′ splice site, increased concentrations of ASF/SF2 promote use of the 5′ site nearest to the 3′ site at the expense of the other site (3316; 3317). This effect of ASF/SF2 can be counteracted by another splicing factor, SF5. The exact molecular roles of the factors in controlling splice utilization are not yet known, but we see in general terms that alternative splicing involving different 5′ sites may be influenced by proteins involved in spliceosome assembly. In the case of T/t antigens, the effect probably rests on increased binding of the SR proteins to the site that is preferentially used. Alternative splicing also may be influenced by repression of one site. Exons 2 and 3 of the mouse troponin T gene are mutually exclusive; exon 2 is used in smooth muscle, but exon 3 is used in other tissues. Smooth muscle contains proteins that bind to repeated elements located on either side of exon 3, and which prevent use of the 3′ and 5′ sites that are needed to include it. The pathway of sex determination in D. melanogaster involves interactions between a series of genes in which alternative splicing events distinguish male and female. The pathway takes the form illustrated in Figure 24.22, in which the ratio of X chromosomes to autosomes determines the expression of sxl, and changes in expression are passed sequentially through the other genes to dsx, the last in the pathway. The pathway starts with sex-specific splicing of sxl. Exon 3 of the sxl gene contains a termination codon that prevents synthesis of functional protein. This exon is included in the mRNA produced in males, but is skipped in females. (Exon skipping illustrated for another example in Figure ) As a result, only females produce Sxl protein. The protein has a concentration of basic amino acids that resembles other RNA-binding proteins. The presence of Sxl protein changes the splicing of the transformer (tra) gene. Figure shows that this involves splicing a constant 5′ site to alternative 3′ sites. One splicing pattern occurs in both males and females, and results in an RNA that has an early termination codon. The presence of Sxl protein inhibits usage of the normal 3′ splice site by binding to the polypyrimidine tract at its branch site (3319). When this site is skipped, the next 3′ site is used. This generates a female-specific mRNA that codes for a protein. So tra produces a protein only in females; this protein is a splicing regulator. tra2 has a similar function in females (but is also expressed in the male germline). The Tra and Tra2 proteins are SR splicing factors that act directly upon the target transcripts. Tra and Tra2 cooperate (in females) to affect the splicing of dsx. Figure shows examples of cases in which splice sites are used to add or to substitute exons or introns, again with the consequence that different protein products are generated. In the doublesex (dsx) gene, females splice the 5′ site of intron 3 to the 3′ site of that intron; as a result translation terminates at the end of exon 4. Males splice the 5′ site of intron 3 directly to the 3′ site of intron 4, thus omitting exon 4 from the mRNA, and allowing translation to continue through exon 6. The result of the alternative splicing is that different proteins are produced in each sex: the male product blocks female sexual differentiation, while the female product represses expression of male-specific genes. Alternative splicing of dsx RNA is controlled by competition between 3′ splice sites. dsx RNA has an element downstream of the leftmost 3′ splice site that is bound by Tra2; Tra and SR proteins associate with Tra2 at the site, which becomes an enhancer that assists binding of U2AF at the adjacent pyrimidine tract (3320; 3321). This commits the formation of the spliceosome to use this 3′ site in females rather than the alternative 3′ site. The proteins recognize the enhancer cooperatively, possibly relying on formation of some secondary structure as well as sequence per se. Sex determination therefore has a pleasing symmetry: the pathway starts with a female-specific splicing event that causes omission of an exon that has a termination codon, and ends with a female-specific splicing event that causes inclusion of an exon that has a termination codon. The events have different molecular bases. At the first control point, Sxl inhibits the default splicing pattern. At the last control point, Tra and Tra2 cooperate to promote the female-specific splice. The Tra and Tra2 proteins are not needed for normal splicing, because in their absence flies develop normally (as males). As specific regulators, they need not necessarily participate in the mechanics of the splicing reaction; in this respect they differ from SF2, which is a factor required for general splicing, but can also influence choice of alternative splice sites. P elements of D. melanogaster show a tissue-specific splicing pattern. In somatic cells, there are two splicing events, but in germline an additional splicing event removes another intron. Because a termination codon lies in the germline-specific intron, a longer protein (with different properties) is produced in germline. We discuss the consequences for control of transposition in P elements are activated in the germline, and note for now that the tissue specificity results from differences in the splicing apparatus. The default splicing pathway of the P element pre-mRNA when the RNA is subjected to a heterologous (human) splicing extract is the germline pattern, in which intron 3 is excised. But extracts of somatic cells of D. melanogaster contain a protein that inhibits excision of this intron. The protein binds to sequences in exon 3; if these sequences are deleted, the intron is excised. The function of the protein is therefore probably to repress association of the spliceosome with the 5′ site of intron 3. 某些外显子可能会由于某一对剪接位点的使用与否而被包含或被剔除于RNA产物中.

23 两个位点参与的可变剪接可能导致外显子的增加或替代.

24 24.13 trans-splicing reactions use small RNAs. 反式剪接反应需要小RNA.反式剪接反应发生于锥虫和蠕虫中, 其中一个小序列(SL RNA)剪接到RNA前体的5'端. SL RNA (Spliced leader RNA) is a small RNA that donates an exon in the trans-splicing reaction of trypanosomes and nematodes. Splicing reactions usually occur only in cis between splice junctions on the same molecule of RNA. trans-splicing occurs in trypanosomes and worms where a short sequence (SL RNA) is spliced to the 5′ ends of many precursor mRNAs. SL RNA has a structure resembling the Sm-binding site of U snRNAs and may play an analogous role in the reaction. In both mechanistic and evolutionary terms, splicing has been viewed as an intramolecular reaction, amounting essentially to a controlled deletion of the intron sequences at the level of RNA. In genetic terms, splicing occurs only in cis. This means that only sequences on the same molecule of RNA can be spliced together. The upper part of Figure shows the normal situation. The introns can be removed from each RNA molecule, allowing the exons of that RNA molecule to be spliced together, but there is no intermolecular splicing of exons between different RNA molecules. We cannot say that trans splicing never occurs between pre-mRNA transcripts of the same gene, but we know that it must be exceedingly rare, because if it were prevalent the exons of a gene would be able to complement one another genetically instead of belonging to a single complementation group. Some manipulations can generate trans-splicing. In the example illustrated in the lower part of Figure 24.24, complementary sequences were introduced into the introns of two RNAs. Base pairing between the complements should create an H-shaped molecule. This molecule could be spliced in cis, to connect exons that are covalently connected by an intron, or it could be spliced in trans, to connect exons of the juxtaposed RNA molecules. Both reactions occur in vitro. Another situation in which trans-splicing is possible in vitro occurs when substrate RNAs are provided in the form of one containing a 5′ splice site and the other containing a 3′ splice site together with appropriate downstream sequences (which may be either the next 5′ splice site or a splicing enhancer). In effect, this mimics splicing by exon definition (see the right side of Figure 24.12), and shows that in vitro it is not necessary for the left and right splice sites to be on the same RNA molecule. These results show that there is no mechanistic impediment to trans-splicing. They exclude models for splicing that require processive movement of a spliceosome along the RNA. It must be possible for a spliceosome to recognize the 5′ and 3′ splice sites of different RNAs when they are in close proximity. Although trans-splicing is rare, it occurs in vivo in some special situations. One is revealed by the presence of a common 35 base leader sequence at the end of numerous mRNAs in the trypanosome. But the leader sequence is not coded upstream of the individual transcription units. Instead it is transcribed into an independent RNA, carrying additional sequences at its 3′ end, from a repetitive unit located elsewhere in the genome. Figure shows that this RNA carries the 35 base leader sequence followed by a 5′ splice site sequence. The sequences coding for the mRNAs carry a 3′ splice site just preceding the sequence found in the mature mRNA (730). When the leader and the mRNA are connected by a trans-splicing reaction, the 3′ region of the leader RNA and the 5′ region of the mRNA in effect comprise the 5′ and 3′ halves of an intron. When splicing occurs, a 5′ – 2′ link forms by the usual reaction between the GU of the 5′ intron and the branch sequence near the AG of the 3′ intron. Because the two parts of the intron are not covalently linked, this generates a Y-shaped molecule instead of a lariat (729). A similar situation is presented by the expression of actin genes in C. elegans. Three actin mRNAs (and some other RNAs) share the same 22 base leader sequence at the 5′ terminus. The leader sequence is not coded in the actin gene, but is transcribed independently as part of a 100 base RNA coded by a gene elsewhere. trans-splicing also occurs in chloroplasts (731). The RNA that donates the 5′ exon for trans splicing is called the SL RNA (spliced leader RNA). The SL RNAs found in several species of trypanosomes and also in the nematode (C. elegans) have some common features. They fold into a common secondary structure that has three stem-loops and a single-stranded region that resembles the Sm-binding site. The SL RNAs therefore exist as snRNPs that count as members of the Sm snRNP class. Trypanosomes possess the U2, U4, and U6 snRNAs, but do not have U1 or U5 snRNAs. The absence of U1 snRNA can be explained by the properties of the SL RNA, which can carry out the functions that U1 snRNA usually performs at the 5′ splice site; thus SL RNA in effect consists of an snRNA sequence possessing U1 function, linked to the exon-intron site that it recognizes. There are two types of SL RNA in C. elegans. SL1 RNA (the first to be discovered) is used for splicing to coding sequences that are preceded only by 5′ nontranslated regions (the most common situation). SL2 RNA is used in cases in which a pre-mRNA contains two coding sequences; it is spliced to the second sequence, thus releasing it from the first, and allowing it to be used as an independent mRNA (732; 733; for review see 250). About 15% of all genes in C. elegans are organized in transcription units that include more than one gene (most often 2-3 genes) (2862). The significance of this form of organization for control of gene expression is not clear. These transcription units do not generally resemble operons where the genes function coordinately in a pathway. The trans-splicing reaction of the SL RNA may represent a step towards the evolution of the pre-mRNA splicing apparatus. The SL RNA provides in cis the ability to recognize the 5′ splice site, and this probably depends upon the specific conformation of the RNA. The remaining functions required for splicing are provided by independent snRNPs. The SL RNA can function without participation of proteins like those in U1 snRNP, which suggests that the recognition of the 5′ splice site depends directly on RNA.

25 剪接通常发生在同一个RNA分子中, 但在另一种情况下, 由于不同RNA内含子之间配对, 能形成一种特殊的结构, 这时会发生反式剪接.

26 锥虫SL RNA的一个外显子可以通过反式剪接与RNA的第一个外显子连接在一起锥虫SL RNA的一个外显子可以通过反式剪接与RNA的第一个外显子连接在一起. 这个反应与细胞核的顺式剪接反应具有同样的相互作用, 但是产生Y型而不是套索结构.

27 24. 14 Yeast tRNA splicing involves cutting and rejoining24.14 Yeast tRNA splicing involves cutting and rejoining. 酵母tRNA剪接包括切割和重连两部分. An RNA ligase is an enzyme that functions in tRNA splicing to make a phosphodiester bond between the two exon sequences that are generated by cleavage of the intron. tRNA splicing occurs by successive cleavage and ligation reactions. Most splicing reactions depend on short consensus sequences and occur by transesterification reactions in which breaking and making of bonds is coordinated. The splicing of tRNA genes is achieved by a different mechanism that relies upon separate cleavage and ligation reactions. Some 59 of the 272 nuclear tRNA genes in the yeast S. cerevisiae are interrupted. Each has a single intron, located just one nucleotide beyond the 3′ side of the anticodon. The introns vary in length from bp. Those in related tRNA genes are related in sequence, but the introns in tRNA genes representing different amino acids are unrelated. There is no consensus sequence that could be recognized by the splicing enzymes. This is also true of interrupted nuclear tRNA genes of plants, amphibians, and mammals. All the introns include a sequence that is complementary to the anticodon of the tRNA. This creates an alternative conformation for the anticodon arm in which the anticodon is base paired to form an extension of the usual arm. An example is drawn in Figure Only the anticodon arm is affected — the rest of the molecule retains its usual structure. The exact sequence and size of the intron is not important. Most mutations in the intron do not prevent splicing. Splicing of tRNA depends principally on recognition of a common secondary structure in tRNA rather than a common sequence of the intron. Regions in various parts of the molecule are important, including the stretch between the acceptor arm and D arm, in the Tψ C arm, and especially the anticodon arm. This is reminiscent of the structural demands placed on tRNA for protein synthesis (see Protein synthesis). The intron is not entirely irrelevant, however. Pairing between a base in the intron loop and an unpaired base in the stem is required for splicing. Mutations at other positions that influence this pairing (for example, to generate alternative patterns for pairing) influence splicing. The rules that govern availability of tRNA precursors for splicing resemble the rules that govern recognition by aminoacyl-tRNA synthetases (see tRNAs are charged with amino acids by synthetases). In a temperature-sensitive mutant of yeast that fails to remove the introns, the interrupted precursors accumulate in the nucleus. The precursors can be used as substrates for a cell-free system extracted from wild-type cells. The splicing of the precursor can be followed by virtue of the resulting size reduction. This is seen by the change in position of the band on gel electrophoresis, as illustrated in Figure The reduction in size can be accounted for by the appearance of a band representing the intron. The cell-free extract can be fractionated by assaying the ability to splice the tRNA. The in vitro reaction requires ATP. Characterizing the reactions that occur with and without ATP shows that the two separate stages of the reaction are catalyzed by different enzymes. The first step does not require ATP. It involves phosphodiester bond cleavage by an atypical nuclease reaction. It is catalyzed by an endonuclease. The second step requires ATP and involves bond formation; it is a ligation reaction, and the responsible enzyme activity is described as an RNA ligase. tRNA剪接经历了连续的切割和重连反应.

28 酵母苯丙氨酰-tRNA前体中的一个内含子可以和反密码子环配对, 从而改变反密码子臂的结构酵母苯丙氨酰-tRNA前体中的一个内含子可以和反密码子环配对, 从而改变反密码子臂的结构. 此tRNA前体内含子 “环” 与 “颈” 上一个受排挤碱基的配对可能是剪接所必须的.

29 24.15 The splicing endonuclease recognizes tRNA. 剪接时核酸内切酶识别tRNA.酵母核酸内切酶是一种有两个(相关)催化亚基组成的异源四聚体. 它使用测量机制来确定切割位点, 其位置与RNA结构中的某一点有关. An endonuclease cleaves the tRNA precursors at both ends of the intron. The yeast endonuclease is a heterotetramer, with two (related) catalytic subunits. It uses a measuring mechanism to determine the sites of cleavage by their positions relative to a point in the tRNA structure. The archaeal nuclease has a simpler structure and recognizes a bulge-helix-bulge structural motif in the substrate. The endonuclease is responsible for the specificity of intron recognition. It cleaves the precursor at both ends of the intron. The yeast endonuclease is a heterotetrameric protein. Its activities are illustrated in Figure The related subunits Sen34 and Sen2 cleave the 3′ and 5′ splice sites, respectively. Subunit Sen54 may determine the sites of cleavage by "measuring" distance from a point in the tRNA structure. This point is in the elbow of the (mature) L-shaped structure (2878). The role of subunit Sen15 is not known, but its gene is essential in yeast. The base pair that forms between the first base in the anticodon loop and the base preceding the 3′ splice site is required for 3′ splice site cleavage (735; 736; 737; 2880). An interesting insight into the evolution of tRNA splicing is provided by the endonucleases of archaea. These are homodimers or homotetramers, in which each subunit has an active site (although only two of the sites function in the tetramer) that cleaves one of the splice sites (2879). The subunit has sequences related to the sequences of the active sites in the Sen34 and Sen2 subunits of the yeast enzyme. However, the archaeal enzymes recognize their substrates in a different way. Instead of measuring distance from particular sequences, they recognize a structural feature, called the bulge-helix-bulge. Figure shows that cleavage occurs in the two bulges (2877; 2876). So the origin of splicing of tRNA precedes the separation of the archaea and the eukaryotes. If it originated by insertion of the intron into tRNAs, this must have been a very ancient event.

30 酿酒酵母tRNA前体的5'和3'的切割是由核酸内切酶的不同亚基催化的酿酒酵母tRNA前体的5'和3'的切割是由核酸内切酶的不同亚基催化的. 另一个亚基可能通过测量成熟tRNA结构中的某个点的距离, 以此为参数决定切割位点的位置. AI碱基对同样很重要.

31 RNA聚合酶I在18个碱基终止序列处终止转录. RNA聚合酶III在G-C富含序列中的poly(U)4序列处终止转录.24.18 The 3′ends of polI and polIII transcripts are generated by termination. 终止反应产生聚合酶I和聚合酶III转录物的3'端. RNA polymerase I terminates transcription at an 18 base terminator sequence. RNA polymerase III terminates transcription in poly(U)4 sequence embedded in a G•C-rich sequence. 3′ ends of RNAs can be generated in two ways. Some RNA polymerases terminate transcription at a defined (terminator) sequence in DNA, as shown in Figure Other RNA polymerases do not show discrete termination, but continue past the site corresponding to the 3′ end, which is generated by cleavage of the RNA by an endonuclease, as shown in Figure Information about the termination reaction for eukaryotic RNA polymerases is less detailed than our knowledge of initiation. RNA polymerases I and III have discrete termination events (like bacterial RNA polymerase), but it is not clear whether RNA polymerase II usually terminates in this way. For RNA polymerase I, the sole product of transcription is a large precursor that contains the sequences of the major rRNA. The precursor is subjected to extensive processing. Termination occurs at a discrete site >1000 bp downstream of the mature 3′ end, which is generated by cleavage. Termination involves recognition of an 18 base terminator sequence by an ancillary factor. With RNA polymerase III, transcription in vitro generates molecules with the same 5′ and 3′ ends as those synthesized in vivo. The termination reaction resembles intrinsic termination by bacterial RNA polymerase (see There are two types of terminators in ). Termination usually occurs at the second U within a run of 4 U bases, but there is heterogeneity, with some molecules ending in 3 or even 4 U bases. The same heterogeneity is seen in molecules synthesized in vivo, so it seems to be a bona fide feature of the termination reaction. Just like the prokaryotic terminators, the U run is embedded in a G•C-rich region. Although sequences of dyad symmetry are present, they are not needed for termination, since mutations that abolish the symmetry do not prevent the normal completion of RNA synthesis. Nor are any sequences beyond the U run necessary, since all distal sequences can be replaced without any effect on termination. The U run itself is not sufficient for termination, because regions of 4 successive U residues exist within transcription units read by RNA polymerase III. (However, there are no internal U5 runs, which fits with the greater efficiency of termination when the terminator is a U5 rather than U4 sequence.) The critical feature in termination must therefore be the recognition of a U4 sequence in a context that is rich in G•C base pairs. How does the termination reaction occur? It cannot rely on the weakness of the rU-dA RNA-DNA hybrid region that lies at the end of the transcript, because often only the first two U residues are transcribed. Perhaps the G•C-rich region plays a role in slowing down the enzyme, but there does not seem to be a counterpart to the hairpin involved in prokaryotic termination. We remain puzzled how the enzyme can respond so specifically to such a short signal. And in contrast with the initiation reaction, which RNA polymerase III cannot accomplish alone, termination seems to be a function of the enzyme itself.  RNA聚合酶I在18个碱基终止序列处终止转录. RNA聚合酶III在G-C富含序列中的poly(U)4序列处终止转录.

32 当终止产生3'端, RNA聚合酶和RNA在DNA的终止子序列上被释放.

33 当核酸内切酶在RNA的特定序列处切割并产生3'端时, RNA聚合酶继续转录.

34 序列AAUAAA是一种切割信号, 用于产生多聚腺苷酸化的mRNA 3'端. 特异性因子和核酸内切酶在AAUAAA下游切割mRNA.24.19 The 3'ends of mRNAs are generated by cleavage and polyadenylation. mRNA的3'端由切割和多聚腺苷酸化产生. 序列AAUAAA是一种切割信号, 用于产生多聚腺苷酸化的mRNA 3'端. 特异性因子和核酸内切酶在AAUAAA下游切割mRNA. 特异性因子和poly(A)聚合酶在3'端连续添加约200个A残基. Cordycepin is 3′ deoxyadenosine, an inhibitor of polyadenylation of RNA. Endonucleases cleave bonds within a nucleic acid chain; they may be specific for RNA or for single-stranded or double-stranded DNA. Poly(A) polymerase is the enzyme that adds the stretch of polyadenylic acid to the 3′ end of eukaryotic mRNA. It does not use a template. The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated. The reaction requires a protein complex that contains a specificity factor, an endonuclease, and poly(A) polymerase. The specificity factor and endonuclease cleave RNA downstream of AAUAAA. The specificity factor and poly(A) polymerase add ~200 A residues processively to the 3′ end. A•U-rich sequences in the 3′ tail control cytoplasmic polyadenylation or deadenylation during Xenopus embryonic development. The 3′ ends of mRNAs are generated by cleavage followed by polyadenylation. Addition of poly(A) to nuclear RNA can be prevented by the analog 3′ – deoxyadenosine, also known as cordycepin. Although cordycepin does not stop the transcription of nuclear RNA, its addition prevents the appearance of mRNA in the cytoplasm. This shows that polyadenylation is necessary for the maturation of mRNA from nuclear RNA. Generation of the 3′ end is illustrated in Figure RNA polymerase transcribes past the site corresponding to the 3′ end, and sequences in the RNA are recognized as targets for an endonucleolytic cut followed by polyadenylation. A single processing complex undertakes both the cutting and polyadenylation. The polyadenylation stabilizes the mRNA against degradation from the 3′ end. Its 5′ end is already stabilized by the cap. RNA polymerase continues transcription after the cleavage, but the 5′ end that is generated by the cleavage is unprotected. The cleavage event provides an indirect trigger for termination by RNA polymerase II. An exonuclease binds to the 5′ end of the RNA that is continuing to be transcribed after cleavage. It degrades the RNA faster than it is synthesized, so that it catches up with RNA polymerase. It then interacts with ancillary proteins that are bound to the CTD of the polymerase, and this interaction triggers the release of RNA polymerase from DNA, causing transcription to terminate (5578). The overall model is similar to that for the role of rho in terminating transcription by bacterial RNA polymerase (see How does rho factor work? ). This explains why the termination sites for RNA polymerase II are not well defined, but may occur at varying locations within a long region downstream of the site corresponding to the 3′ end of the RNA. A common feature of mRNAs in higher eukaryotes (but not in yeast) is the presence of the highly conserved sequence AAUAAA in the region from nucleotides upstream of the site of poly(A) addition. Deletion or mutation of the AAUAAA hexamer prevents generation of the polyadenylated 3′ end. The signal is needed for both cleavage and polyadenylation (744; 745; for review see 248). The development of a system in which polyadenylation occurs in vitro opened the route to analyzing the reactions. The formation and functions of the complex that undertakes 3′ processing are illustrated in Figure Generation of the proper 3′ terminal structure requires an endonuclease (consisting of the components CFI and CFII) to cleave the RNA, a poly(A) polymerase (PAP) to synthesize the poly(A) tail, and a specificity component (CPSF) that recognizes the AAUAAA sequence and directs the other activities. A stimulatory factor, CstF, binds to a G-U-rich sequence that is downstream from the cleavage site itself (746). The specificity factor contains 4 subunits, which together bind specifically to RNA containing the sequence AAUAAA. The individual subunits are proteins that have common RNA-binding motifs, but which by themselves bind nonspecifically to RNA. Protein-protein interactions between the subunits may be needed to generate the specific AAUAAA-binding site. CPSF binds strongly to AAUAAA only when CstF is also present to bind to the G-U-rich site. The specificity factor is needed for both the cleavage and polyadenylation reactions. It exists in a complex with the endonuclease and poly(A) polymerase, and this complex usually undertakes cleavage followed by polyadenylation in a tightly coupled manner. The two components CFI and CFII (cleavage factors I and II), together with specificity factor, are necessary and sufficient for the endonucleolytic cleavage. The poly(A) polymerase has a nonspecific catalytic activity. When it is combined with the other components, the synthetic reaction becomes specific for RNA containing the sequence AAUAAA. The polyadenylation reaction passes through two stages. First, a rather short oligo(A) sequence (~10 residues) is added to the 3′ end. This reaction is absolutely dependent on the AAUAAA sequence, and poly(A) polymerase performs it under the direction of the specificity factor. In the second phase, the oligo(A) tail is extended to the full ~200 residue length. This reaction requires another stimulatory factor that recognizes the oligo(A) tail and directs poly(A) polymerase specifically to extend the 3′ end of a poly(A) sequence. The poly(A) polymerase by itself adds A residues individually to the 3′ position. Its intrinsic mode of action is distributive; it dissociates after each nucleotide has been added. However, in the presence of CPSF and PABP (poly(A)-binding protein), it functions processively to extend an individual poly(A) chain. The PABP is a 33 kD protein that binds stoichiometrically to the poly(A) stretch. The length of poly(A) is controlled by the PABP, which in some way limits the action of poly(A) polymerase to ~200 additions of A residues. The limit may represent the accumulation of a critical mass of PABP on the poly(A) chain. PABP binds to the translation initiation factor eIF4G, thus generating a closed loop in which a protein complex contains both the 5′ and 3′ ends of the mRNA (see Figure 6.20 in Eukaryotes use a complex of many initiation factors). Polyadenylation is an important determinant of mRNA function. It may affect both stability and initiation of translation (see The 3 terminus is polyadenylated). In embryonic development in some organisms, the presence of poly(A) is used to control translation, and preexisting mRNAs may either be polyadenylated (to stimulate translation) or deadenylated (to terminate translation). During Xenopus embryonic development, polyadenylation of mRNA in the cytoplasm in Xenopus depends on a specific cis-acting element (the CPE) in the 3′ tail. This is another AU-rich sequence, UUUUUAU (2313; 2314). In Xenopus embryos at least two type of cis-acting sequences found in the 3′ tail can trigger deadenylation. EDEN (embryonic deadenylation element) is a 17 nucleotide sequence (2310). ARE elements are AU-rich, usually containing tandem repeats of AUUUA (2311). There is a poly(A)-specific RNAase (PARN) that could be involved in the degradation (2312). Of course, deadenylation is not always triggered by specific elements; in some situations (including the normal degradation of mRNA as it ages), poly(A) is degraded unless it is specifically stabilized. 

35 序列AAUAAA是切割产生3'端和多聚腺苷酸化所必需的.

36 3'端加工复合体具有多种活性, 每个CPSF和CF都有数个亚基, 其余组分为单体.

37 组蛋白mRNA不被多聚腺苷酸化, 它们的3'端由切割反应产生, 它依赖于mRNA的结构. 24.20 Cleavage of the 3′ end of histone mRNA may require a small RNA.  组蛋白mRNA的3'端切割可能需要小RNA. Histone mRNAs are not polyadenylated; their 3′ ends are generated by a cleavage reaction that depends on the structure of the mRNA. The cleavage reaction requires the SLBP to bind to a stem-loop structure, and the U7 snRNA to pair with an adjacent single-stranded region. Some mRNAs are not polyadenylated. The formation of their 3′ ends is therefore different from the coordinated cleavage/polyadenylation reaction. The most prominent members of this mRNA class are the mRNAs coding for histones that are synthesized during DNA replication. Formation of their 3′ ends depends upon secondary structure. The structure at the 3′ terminus is a highly conserved stem-loop structure, with a stem of 6 bp and a loop of 4 nucleotides. Cleavage occurs 4-5 bases downstream of the stem-loop. Two factors are required for the cleavage reaction: the stem-loop binding protein (SLBP) recognizes the structure (3324); and the U7 snRNA pairs with a purine-rich sequence (the histone downstream element, or HDE) located ~10 nucleotides downstream of the cleavage site (743; 3325). Mutations that prevent formation of the duplex stem of the stem-loop prevent formation of the end of the RNA. Secondary mutations that restore duplex structure (though not necessarily the original sequence) behave as revertants. This suggests that formation of the secondary structure is more important than the exact sequence. The SLBP binds to the stem-loop and then interacts with U7 snRNP to enhance its interaction with the downstream binding site for U7 snRNA (3326). U7 snRNP is a minor snRNP consisting of the 63 nucleotide U7 snRNA and a set of several proteins (including Sm proteins; see snRNAs are required for splicing). The reaction between histone H3 mRNA and U7 snRNA is drawn in Figure The upstream hairpin and the HDE that pairs with U7 snRNA are conserved in histone H3 mRNAs of several species. The U7 snRNA has sequences towards its 5′ end that pair with the histone mRNA consensus sequences. 3′ processing is inhibited by mutations in the HDE that reduce ability to pair with U7 snRNA. Compensatory mutations in U7 snRNA that restore complementarity also restore 3′ processing (3327). This suggests that U7 snRNA functions by base pairing with the histone mRNA. The sequence of the HDE varies among the various histone mRNAs, with the result that binding of snRNA is not by itself necessarily stable, but requires also the interaction with SLBP Cleavage to generate a 3′ terminus occurs a fixed distance from the site recognized by U7 snRNA, which suggests that the snRNA is involved in defining the cleavage site (for review see 241). However, the factor(s) actually responsible for cleavage have not yet been identified.  组蛋白mRNA不被多聚腺苷酸化, 它们的3'端由切割反应产生, 它依赖于mRNA的结构. 切割反应需要SLBP结合到茎环结构, 以及需要U7 snRNA与相邻单链区配对.

38 组蛋白H3 mRNA 3'端的产生取决于3'端一个保守的发夹结构和一段能和U7 snRNA配对的序列.

39 24.21 Production of rRNA requires cleavage events. rRNA的产生需要切割反应.45S RNA is a precursor that contains the sequences of both major ribosomal RNAs (28S and 18S rRNAs). The large and small rRNAs are released by cleavage from a common precursor RNA. The major rRNAs are synthesized as part of a single primary transcript that is processed to generate the mature products. The precursor contains the sequences of the 18S, 5.8S, and 28S rRNAs. In higher eukaryotes, the precursor is named for its sedimentation rate as 45S RNA. In lower eukaryotes, it is smaller (35S in yeast). The mature rRNAs are released from the precursor by a combination of cleavage events and trimming reactions (for review see 980). Figure shows the general pathway in yeast. There can be variations in the order of events, but basically similar reactions are involved in all eukaryotes. Most of the 5′ ends are generated directly by a cleavage event. Most of the 3′ ends are generated by cleavage followed by a 3′ – 5′ trimming reaction. Many ribonucleases have been implicated in processing rRNA, including the exosome, an assembly of several exonucleases that also participates in mRNA degradation (see mRNA degradation involves multiple activities). Mutations in individual enzymes usually do not prevent processing, suggesting that their activities are redundant and that different combinations of cleavages can be used to generate the mature molecules. There are always multiple copies of the transcription unit for the rRNAs. The copies are organized as tandem repeats (see The repeated genes for rRNA maintain constant sequence). 5S RNA is transcribed from separate genes by RNA polymerase III. Usually the 5S genes are clustered, but are separate from the genes for the major rRNAs. (In the case of yeast, a 5S gene is associated with each major transcription unit, but is transcribed independently.) There is a difference in the organization of the precursor in bacteria. The sequence corresponding to 5.8S rRNA forms the 5′ end of the large (23S) rRNA, that is, there is no processing between these sequences. Figure shows that the precursor also contains the 5S rRNA and one or two tRNAs. In E. coli, the 7 rrn operons are dispersed around the genome; four rrn loci contain one tRNA gene between the 16S and 23S rRNA sequences, and the other rrn loci contain two tRNA genes in this region. Additional tRNA genes may or may not be present between the 5S sequence and the 3′ end. So the processing reactions required to release the products depend on the content of the particular rrn locus. In both prokaryotic and eukaryotic rRNA processing, ribosomal proteins (and possibly also other proteins) bind to the precursor, so that the substrate for processing is not the free RNA but is a ribonucleoprotein complex. 大小rRNA都通过从共同的RNA前体切割下来而释放.

40 真核生物的成熟rRNA是由初始转录物经切割和修整反应而产生.

41 大肠杆菌中rrn操纵子包含rRNA和tRNA基因, 转录物准确的长度取决于使用哪个启动子(P)和终止子(t), 每个RNA产物必需从转录物的任一端被切割而释放.

42 复习题 内含子的主要结构? 内含子剪接的主要步骤? 什么是差异剪接? mRNA 3'端的形成?