分子生物学教学课件：CHAPTER 13 RNA splicing

1、Recall the structuer of RNAtRNArRNAFigure 13-1Primary transcriptThe number of introns per gene in various eukaryotic speciesFigure 13-2Try to find out the answer after finish the Try to find out the answer after finish the study of this chapter!study of this chapter!OutlineThe chemistry of RNA splic

2、ingFigure 13-2The consensus sequences for humann 5splice site (5剪接位点剪接位点): the exon-intron boundary at the 5 end of the intron.n 3 splice site (3剪接位点剪接位点): the exon-intron boundary at the 3 end of the intron.n Branch point site (分枝位点分枝位点): an A close to the 3 end of the intron, which is followed by

3、a polypyrimidine tract (Py tract).Two successive transesterification:Three-way junction Figure 13-4Lariat FormationThe structure of three-way functionFigure 13-5Branch site is a 3-way junction including 2OH creating a “branch” point.Figure 13-4Lariat FormationTrans-splicingFigure 13-6Not a lariat Ex

4、ons dont have to be spliced to the next exon in the pre-mRNA. In alternative splicing exons can be skipped. In some cases, an exon from one RNA molecule can be spliced to an exon in another molecule = trans-splicing. Trans-splicing is rare, but can be common in some organisms (trypanosomes and C. el

5、egans). RNA splicing is carried out in a large protein/RNA complex called a spliceosome = 150 proteins and 5 RNAs. Many functions of the spliceosome are carried out by its RNA components. Activity of the spliceosome requires ATP hydrolysis.SpliceosomeFive RNAs (U1, U2, U4, U5, and U6, 100-300 nt)The

6、 spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reactionFigure 13-6RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA Assembly Rearrangement CatalysisE complexA complexStep 1Step 2Figure 13-8 A complexB complexFigure 13-

7、8 Figure 13-8Figure 13-6cB complexC complex in which the catalysis has not occurred yetCatalysis Step 2:nU5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3-OH of the 5 exon attacks the 3 splice site.Final Step: Release of the mRNA product

8、 and the snRNPs.Figure 13-8C complex splicesome-mediated splicing reactionsFigure 13-8E complexA complexB complexsplicesome-mediated splicing reactionsSelf-splicing intronGroup I intronsG instead of Aa linear introna Lariat intronFigure 13-9Figure 13-10The similarity of the structures of group II in

9、trons and U2-U6 snRNA complex formed to process first transesterificationFigure 13-9Two kinds of splice-site recognition errorsnSplice sites can be skipped.n“Pseudo” splice sites could be mistakenly recognized, particularly the 3 splice site. Figure 13-11 Recognition is prone to 2 types of errors: E

10、xon skipping join 5 to 3 of wrong exon Mistakenly identifying seq. as a splice site = pseudo splice sight this is particularly easy b/c consensus seq.s are looseReasons for the recognition errors(2) The splice site consensus sequence are rather loose. For example, only AG G tri-nucleotides is requir

11、ed for the 3 splice site, and this consensus sequence occurs every 64 nt theoretically. Two ways to enhance the accuracy of the splice-site selectionSR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites. Fig

12、ure 13-12SR proteins are essential for splicingOutlineAlternative splicingDrosophila DSCAM gene can be spliced in 38,000 alternative ways Figure 13-19Figure 13-15nConstitutive alternative splicing: more than one product is always made from a pre-mRNAnRegulative alternative splicing: different forms

13、of mRNA are produced at different time, under different conditions, or in different cell or tissue typesAn example of constitutive alternative splicing : Splicing of the SV40 T antigen RNAFigure 13-16Regulated alternative splicingFigure 13-22Binds at each end of the exon and conceals (隐藏隐藏) it Coats

14、 the RNA and makes the exons invisible to the splicing machineryAn example of repressors: inhibition of splicing by hnRNPIFigure 13-23Figure 13-13The AT-AC spliceosomeU11 and U12 are in places of U1 and U2, respectivelyOutline5-1 Exons are shuffled by recombin-ation to produce gene encoding new prot

15、einsFor example: DNA-binding protein Figure 13-26Exons have been reused in genes encoding different proteinsFigure 13-27OutlineI. Site specific deaminationFigure 13-30The human apolipoprotein geneStop codeIn liverIn intestinesStop codeFigure 13-29gRNAsFigure 13-31Outline Movement from the nucleus to

16、 the cytoplasm is an active and carefully regulated process.n The damaged, misprocessed and liberated introns are retained in the nucleus and degraded.nA typical mature mRNA carries a collection of proteins that identifies it as being ready for transport. nExport takes place through the nuclear pore

17、 complex. nOnce in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.Figure 13-321. Why RNA splicing is important? 2. Chemical reaction: determination of the splice sites, the products, trans-splicing3. Spliceosome: splicing pathway and finding the splice sites4. Self-splicing int