页数：51页文件大小：1.02 MB来源： 分子生物学基因Geneppt课件
本课件为《分子生物学》第22章核内剪接与RNA加工（Nuclear splicing），依据 Gene VII 教材编排，沿Nuclear splice等小节推进，讲解本章核心概念与分子机制，配逐页图示与文字版。
Chapter 22 Nuclear splicing
RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.pre-mRNAheterogeneous nuclear RNA(hnRNA). hnRNP
Figure 22.1 hnRNA exists as a ribonucleoprotein particle organized as a series of beads.
Figure 22.2 RNA is modified in the nucleus by additions to the 5′and 3′ends and by splicing to remove the introns. The splicing event requires breakage of the exon-intron junctions and joining of the ends of the exons; the expanded illustr at ion shows the principle schematically, but not the actual order of events. Mature mRNA is transported through nuclear pores to the cytoplasm, where it is translated.
Figure 22.3 The ends of nuclear introns are defined by the GT-AG rule.
Figure 22.4 Splicing junctions are recognized only in the correct pairwise combin at ions.
Figure 2.20 A special splicing vector is used for exon trapping. If an exon is present in the genomic fragment, its sequence will be recovered in the cytoplasmic RNA, but if the genomic fragment consists solely of an intron,.
Figure 22.5 Northern blotting of nuclear RNA with an ovomucoid probe identifies discrete precursors to mRNA. The contents of the more prominent bands are indicated. Photograph kindly provided by Bert O'Malley.
Lariat is an intermediate in RNA splicing in which a circular structure with a tail is created by a 5′-2′ bond.
Figure 22.6 Splicing occurs in two stages, in which the 5′exon is separated and then is joined to the 3′exon.
Figure 22.7 Nuclear splicing occurs by two transesterific at ion reactions in which a free OH end attacks a phosphodiester bond.
scRNAis any one of several small cytoplasmic RNA molecules present in the cytoplasm and (sometimes) nucleus.snRNA(small nuclear RNA) is any one of many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions.
Figure 22.8 U1 snRNA has a base paired structure that creates several domains. The 5′end remains single stranded and can base pair with the 5′splicing site.
Figure 22.9 Mutations that abolish function of the 5′splicing site can be suppressed by compensating mutations in U1 snRNA that restore base pairing.
Figure 22.10 The splicing reaction proceeds through discrete stages in which spliceosome formation involves the interaction of components that recognize the consensus sequences.
Figure 22.11 There may be multiple routes for initial recognition of 5′and 3′splice sites.
Figure 22.12 U6-U4 pairing is incompatible with U6-U2 pairing. When U6 joins the spliceosome it is paired with U4. Release of U4 allows a conformational change in U6; one part of the released sequence forms a hairpin (dark grey), and the other part (black) pairs with U2. Because an adjacent region of U2 is already paired with the branch site, this brings U6 into juxt a position with the branch. Note that the substrate RNA is reversed from the usual orientation and is shown 3′-5′.
Figure 22.13 Splicing utilizes a series of base pairing reactions between snRNAs and splice sites.
Figure 22.17 Nuclear splicing and group II splicing involve the formation of similar secondary structures. The sequences are more specific in nuclear splicing; group II splicing uses positions that may be occupied by either purine (R) or either pyrimidine (Y).
Figure 22.14 Spliceosomes are ellipsoidal particles with several discrete regions. The bar is 50 nm. Photograph kindly provided by Tom Maniat is.
Figure 22.15 Three classes of splicing reactions proceed by two transesterific at ions. First, a free OH group attacks the exon 1 -intron junction. Second, the OH created at the end of exon 1 attacks the intron -exon 2 junction.
Figure 22.6 Splicing occurs in two stages, in which the 5′exon is separated and then is joined to the 3′exon.
Figure 22.16 Splicing releases mitochondrial group II introns in the form of stable lariats. Photograph kindly provided by Leslie Grivell and Annika Arnberg.
Figure 22.17 Nuclear splicing and group II splicing involve the formation of similar secondary structures. The sequences are more specific in nuclear splicing; group II splicing uses positions that may be occupied by either purine (R) or either pyrimidine (Y).
Figure 22.18 Alternative forms of splicing may generate a variety of protein products from an individual gene. Changing the splice sites may introduce termination codons (shown by asterisks) or change reading frames.
Figure 22.10 The splicing reaction proceeds through discrete stages in which spliceosome formation involves the interaction of components that recognize the consensus sequences.
Figure 22.19 Sex determ in at ion in D. melanogaster involves a pathway in which different splicing events occur in females. Blocks at any stage of the pathway result in male development.
Figure 22.20 Alternative splicing events that involve both sites may cause exons to be added or substituted.
Figure 22.21 Splicing usually occurs only in cis between exons carried on the same physical RNA molecule, but trans splicing can occur when special constructs are made that support base pairing between introns.
Figure 22.11 There may be multiple routes for initial recognition of 5′and 3′splice sites.
Figure 22.22 The SL RNA provides an exon that is connected to the first exon of an mRNA by trans-splicing. The reaction involves the same interactions as nuclear cis-splicing, but generates a Y-shaped RNA instead of a lariat.
Figure 22.22 The SL RNA provides an exon that is connected to the first exon of an mRNA by trans-splicing. The reaction involves the same interactions as nuclear cis-splicing, but generates a Y-shaped RNA instead of a lariat.
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Figure 22.23 The intron in yeast tRNAPhebase pairs with the anticodon to change the structure of the anticodon arm. Pairing between an excluded base in the stem and the intron loop in the precursor may be required for splicing.
Figure 22.24 Splicing of yeast tRNA in vitro can be followed by assaying the RNA precursor and products by gel electrophoresis.
Figure 22.25 The 3′and 5′cleavages in S. cerevisiaepre-tRNA are catalyzed by different subunits of the endonuclease. Another subunit may determine location of the cleavage sites by measuring distance from the mature structure. The AI base pair is also important.
Figure 22.26 Splicing of tRNA requires separate nuclease and ligase activities. The exon-intron boundaries are cleaved by the nuclease to generate 2′-3′cyclic phosphate and 5′OH termini. The cyclic phosphate is opened to generate 3′-OH and 2′phosphate groups. The 5′-OH is phosphorylated. After releasing the intron, the tRNA half molecules fold into a tRNA-like structure that now has a 3′-OH, 5′-P break. This is sealed by a ligase.
Figure 22.27 The unfolded protein response occurs by activating special splicing of HAC1 mRNA to produce a transcription factor that recognizes the UPR.
Figure 22.28 When a 3′end is generated by termination, RNA polymerase and RNA are released at a discrete (terminator) sequence in DNA.
Figure 22.29 When a 3′end is generated by cleavage, RNA polymerase continues transcription while an endonuclease cleaves at a defined sequence in the RNA.
Figure 22.30 The sequence AAUAAA is necessary for cleavage to generate a 3′end for polyadenyl at ion.
Figure 22.31 The 3′processing complex consists of several activities. CPSF and CstF each consist of several subunits; the other components are monomeric. The total mass is >900 kD.
Figure 22.32 Gener at ion of the 3′end of histone H3 mRNA depends on a conserved hairpin and a sequence that base pairs with U7 snRNA.
Figure 22.33 Mature rRNAs are generated by cleavage and trimming events from a primary transcript.
Figure 22.34 The rrnoperons contain genes for both rRNA and tRNA. The exact lengths of the transcripts depend on which promoters (P) and terminators (t) are used. Each RNA product must be released from the transcript by cuts on either side.
Figure 22.35 A snoRNA base pairs with a region of rRNA that is to be methylated.
Figure 22.36 An ACA group snoRNA base pairs with rRNA to determine the position of pseudouridine modification.
1.Splicing accomplishes the removal of introns and the joining of exons into the mature sequence of RNA.2.Nuclear splicing follows preferred but not obligatory pathways.3.Nuclear splicing requires formation of a spliceosome, a large particle that assembles the consensus sequences into a reactive conform at ion. 4.Splicing is usually intr a molecular, but some cases have been found of trans-(intermolecular) splicing.
Summary
5.Group II introns share with nuclear introns the use of a lariat as intermediate, but are able to per form the reaction as a self-catalyzed property of the RNA. 6.Yeast tRNA splicing involves separate endonuclease and ligase reactions. 7.The termination capacity of RNA polymerase II has not been characterized, and 3 ends of its transcripts are generated by cleavage.
Summary
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