1. The mechanism of splicing of nuclear mRNA precursors Chapter 14

2. Evidence for Split Genes Most higher eukaryotic genes coding for mRNA and tRNA are interrupted by unrelated regions called introns Exons are present surrounding the introns Exons contain the sequences that finally appear in the mature RNA product –Genes for mRNAs have been found with anywhere from 0 to 362 exons –tRNA genes have either 0 or 1 exon

3. How do introns not find its way into mature RNA products of the genes? - RNA Splicing Introns are never transcribed –Polymerase somehow jumps from one exon to another Introns are transcribed –Primary transcript result- an overlarge gene product is cut down by removing introns –This is correct process

4. RNA splicing Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing Introns are transcribed along with exons in the primary transcript Introns are removed as the exons are spliced together

5. Stages of RNA Splicing Messenger RNA synthesis in eukaryotes occurs in stages First stage: –Synthesis of primary transcript product –This is an mRNA precursor containing introns copied from the gene if present –Precursor is part of a pool of large nuclear RNAs – hnRNAs Second stage: –mRNA maturation –Removal of introns in a process called splicing

6. Splicing Signals Splicing signals in nuclear mRNA precursors are remarkably uniform (exon/GU-intron-AG/exon) –First 2 bases of introns are GU –Last 2 are AG 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs Whole consensus sequences are important to proper splicing (Look at mammalian and yeast consensus sequences on page 403) Abnormal splicing can occur when the consensus sequences are mutated

7. Mechanism of Splicing of Nuclear mRNA Precursors Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat 2-step model –2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1 st exon and G beginning of intron Forms loop of the lariat Separates first exon from intron –3’-OH left at end of 1 st exon attacks phosphodiester bond linking intron to 2 nd exon Forms the exon-exon phosphodiester bond Releases intron in lariat form at same time

8. Simplified Mechanism of Splicing

9. Spliceosomes Splicing takes place on a particle called a spliceosome Yeast and mammalian spliceosomes have sedimentation coefficients of 40S and 60S Spliceosomes contain the pre-mRNA –Along with snRNPs and protein splicing factors –These recognize key splicing signals and orchestrate the splicing process

10. snRNPs Small nuclear RNAs coupled to proteins are abbreviated as snRNPs - small nuclear ribonuclear proteins The snRNAs (small nuclear RNAs) can be resolved on a gel: –U1, U2, U4, U5, U6 –All 5 snRNAs join the spliceosome to play crucial roles in splicing

11. U1 snRNP U1 snRNA sequence is complementary to 5’- splice site consensus sequences –U1 snRNA base-pairs with these splice sites Splicing involves a branch within the intron

12. U6 snRNP U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA Occurs first prior to formation of lariat intermediate U6 also associates with U2 during splicing

13. U2 snRNP U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint U2 also forms base pairs with U6 –This region is called helix I –Helps orient snRNPs for splicing 5’-end of U2 interacts with 3’-end of U6 –This interaction forms a region called helix II –This region is important in splicing in mammalian cells, not in yeast cells

14. U5 snRNP U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon This should result in the two exons lining up for splicing

15. snRNP Involvement in mRNA Splicing Spliceosomal complex contains: –Substrate –U2 –U5 –U6 –All snRNP are made up of same seven set of proteins called Sm proteins

16. Spliceosome Assembly and Function Spliceosome is composed of many components – proteins and RNA These components assemble stepwise The spliceosome cycle: –Assembly –Function –Disassembly By controlling assembly of the spliceosome - a cell can regulate quality and quantity of splicing and so regulate gene expression

17. Spliceosome Cycle Assembly begins with binding of U1 to splicing substrate forming a commitment complex - a unit committed to splicing out the intron U2 joins the complex next - followed by the others U2 binding requires ATP U6 dissociates from U4 and displaces U1 at the 5’- splice site –This step is ATP-dependent –Activates the spliceosome –Allows U1 and U4 to be released

18. Commitment Commitment to splice at a given site is determined by an RNA-binding protein This protein binds to splicing substrate and recruits other spliceosomal components The first component to follow is U1

19. Yeast Two-Hybrid Assay

20. Intron-Bridging Protein-Protein Interactions Branchpoint bridging protein binds to U1 snRNP protein Comparison of yeast to mammalian complexes is seen at right

21. Role of the RNA Polymerase II CTD CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing (figure 14.37) Questions 27, 28 and 31 - Homework

22. Alternative Splicing Transcripts of many eukaryotic genes are subject to alternative splicing –This splicing can have profound effects on the protein products of a gene –Can make a difference between: Secreted or membrane-bound protein Activity and inactivity

23. Alternative Splicing Patterns-Pg 432 Alternative splicing of the same pre-mRNA gives rise to very different products –Alternative splicing patterns occur in over half of human genes –Many genes have more than 2 splicing patterns - some have thousands

24. What stimulates recognition of signals under only some circumstances? - Silencing of Splicing Exons can contain sequences – –Exonic splicing enhancers (ESEs) stimulate splicing –Exonic splicing silencers (ESSs) inhibit splicing

25. Self-Splicing RNAs Some RNAs could splice themselves without aid from a spliceosome or any other protein Tetrahymena 26S rRNA gene has an intron, splices itself in vitro –Group I introns are a group of self-splicing RNAs –Group II introns also have some self-splicing members

26. Group I Introns Group I introns can be removed in vitro with no help from protein Reaction begins with attack by a guanine nucleotide on the 5’-splice site –Adds G to the 5’-end of the intron –Releases the first exon

27. Linear Introns Second step- first exon attacks the 3’-splice site –Ligates 2 exons together –Releases the linear intron Intron cyclizes twice- losing nucleotides each time - then linearizes a last time

28. Group II Introns RNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate - like spliceosome lariats

29. Types of Alternative Splicing Begin transcripts at alternative promoters Some exons can simply be ignored resulting in deletion of the exon Alternative 5’-splice sites can lead to inclusion or deletion of part of an exon Alternative 3’-splice sites can lead to inclusion or deletion of part of an exon A retained intron can be retained in the mRNA if it is not recognized as an intron Polyadenylation causes cleavage of pre-mRNA and loss of downstream exons

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