Bio 402/502 Section II, Lecture 3

1. Bio 402/502Section II, Lecture 3 Transcription & mRNA splicing Dr. Michael C. Yu • Lectures powerpoint files & readings (in PDF files) are located in http://biology.buffalo.edu/Faculty/Yu/yu.html • Office hours: Weds & Thurs, 9am-12pm, or by appt (email for appointment)

2. Transcriptional elongation Steps leading to transcriptional activation CTD phosphorylation status of RNA pol II Promoter escape/clearance Transition to elongation phase (Orphanides & Reinberg, 2000)

3. What happens during transcriptional elongation? • Original contacts within pre-initiation complex abolished • Formation of new contacts with elongation factors • Change of RNA pol II to a ternary complex = high stability • Phosphorylation of CTD (Orphanides & Reinberg, 2000)

4. Model of nucleosome dynamics during transcription • Phosphorylation of the CTD defines the stage of transcription • CTD consists of heptad repeats of the consensus sequence: YSPTSPS CTD: Not phosphorylated • # of repeats differ in organisms • Promoter clearance: Ser #5 gets phosphorylated CTD: phosphorylated • Transition to elongation: Ser #2 gets phosphorylated (Workman, 2006)

5. Experimental evidence for elongation factors • Comparison of RNAPII elongation rate • in vitro: 100-300 nt/min, frequent pauses, and sometimes full arrest • in vivo: 1200-2000 nt/min Why the discrepancy? • Use of pharmacological agents • DRB(5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole • DRB, nucleotide-analogue, cause inhibition of hnRNA transcription by arresting RNA pol II in vivo, but not purified RNA pol II. Possible target? These evidence suggest existence of factors that facilitate transcriptional elongation

6. Biochemical purification identified elongation factors In vitro transcription assays pTEFb TF-IIS Elongin Mechanisms by which elongation factors work: • Elongating through chromatin (such as chromatin remodelers). Ex = SWI/SNF, FACT • Suppression of RNA pol II pausing, ex=elongin, TF-IIF • Liberating RNA pol II from transcriptional arrest, ex = TF-IIS

7. RNA polymerase II often encounters pauses & arrests • Arrest (irreversible backsliding 7-14 nts) • Pause (back-tracking 2-4 nts) • Function of elongation factors: minimize these pauses & arrests (Sims et al, 2004)

8. HIV virus can transactivate by hijacking elongation machinery P-TEFb phosphorylates RNA polII CTD Tat: HIV’s own elongation factor (Karn, HIV database) HIV can bypass pre-initiation complex and head straight for elongation by hijacking RNA pol II from host

9. Nascent RNAs are processed co-transcriptionally • During the message production, processing takes place simultaneously • What are the common mRNA processing events? • Capping • Splicing • 3’-end cleavage/processing • Polyadenylation (Lewin, Genes IX)

10. Capping of pre-mRNAs • Cap=modified guanine nucleotide • Capping= first mRNA processing event - occurs during transcription • CTD recruits capping enzyme as soon as it is phosphorylated • Pre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bp long • Cap structure is recognized by CBC • stablize the transcript • prevent degradation by exonucleases • stimulate splicing and processing

11. Enzymes involved in mRNA capping 2 1 (Proudfoot et al; 2002) 3 1. RNA 5’-triphosphatase (RTP): removal of a single phosphate 2. Guanylyl transferase (GT) - attaches GMP (guanosine 5’-monophosphate) 3. 7-methyltransferase (MT): modifies terminal guanosine

12. Purpose of pre-mRNA capping (Proudfoot et al; 2002) • Protects mRNA from ribonucleases • Distinguish mRNAs from other RNAs • Directs mRNA for transport • Promotes efficient translation • Aid in interaction with transcription machineries

13. Processing of pre-mRNAs Cont’d - splicing • a typical eukaryotic gene has ~4 introns/kb (www.wisc.edu/pharm) • Why splicing? • Multiple proteins from a single gene (alternative splicing) • Facilitate evolution of new genes (“exon shuffling”) (McKee & Silver, 2007)

14. Splicing factors are co-transcriptionally recruited • EM evidence Why co-transcriptional? • Efficiency (zero vs. first order reaction) • Specificity (Lei & Silver, 2004)

15. Mechanism of pre-mRNA splicing branch-point adenosine 5’ splice site 3’ splice site Exon 1 Intron Exon 2 5’- -3’ pre-mRNA trans-esterification 2’ Cut at 5’ site, lariat formation 3’ Cut at 3’ site, exon joining, lariat release trans-esterification ligated exons lariat intron 3’ (www.wisc.edu/pharm)

16. Splice sites are short consensus sequences 5’ splice site 3’ splice site Branch point sequence (Bp) polypyrimidine tract (Py) exon intron intron exon (www.wisc.edu/pharm) • The bigger the nucleotide = more frequent it appears at that position • Black-colored nucleotides are thought to be involved in intron recognition • Splice sites are not always conformed to this consensus

17. Spliceosome assembly is a step-wise event U1 initates splicing by binding to 5’-splice site complex complex U2 binds branch pt C1. 5’-site cleaved & lariat formed C2. 3’-site cleaved complex complex

18. 5 small nuclear RNAs (snRNAs) participate in pre-mRNA splicing U5 U1 U2 U6 orange-interaction with the 5’ splice site green-interaction with the branch site blue-interaction between U2 and U6 tan-Sm-binding site (PuAU4-6GPu) flanked by two stem-loop structures U4 (www.wisc.edu/pharm)

19. Experimental support for the requirement of snRNA in splicing A radiolabeled pre-mRNA is incubated in a nuclear extract in the presence of ATP Reactions are deproteinized and isolated RNA is fractionated on a denaturing polyacrylamide gel Result: Nuclear extracts are competent for splicing a pre-mRNA and the reaction intermediates and products can be visualized after electrophoresis 1. Similar reactions are carried out in the presence of RNaseH (which cuts the RNA strand of a RNA:DNA hybrid) and a DNA oligonucleotide that is complementary to a specific snRNA 2. Examine whether the loss of the snRNA affects production of reaction products or intermediates intermediate lariat Pre-mRNA Spliced product DNA oligo GTTCACATCATCGACA-5’ CAAGUGUAGUAGCUGU RNaseH (www.wisc.edu/pharm)

20. Sm proteins assembles with U-snRNAs (Kambach, C. et al. 1999) • These core snRNP proteins are called Sm because of their reactivity with • antibodies of the Sm serotype from patients with systemic lupus erythematosus • Sm proteins play a key role in hypermethylation of the m7G snRNA cap to m3G, • 3’ end maturation, and nuclear import of the assembled snRNP (www.wisc.edu/pharm)

21. SR proteins play important role in pre-mRNA splicing SR proteins RRM X RS RNA recognition motif arginine/serine-rich domain exon-dependent functions exon-independent functions (SR proteins bind to exon sequences and enhance splicing of the adjacent intron) regulated 3’ splice site selection regulated 5’ splice site selection (U2AF65 binds the polypyrimidine tract) (Graveley, 2000) Facilitate U-snRNP interactions splicing enhancer (www.wisc.edu/pharm)

22. Some lower eukaryotes employ a different type of splicing (www.wisc.edu/pharm) • 13-15% of all genes in C. elegans are expressed as part of an operon

23. branchpoint polypyrimidine tract 5’ splice site TGCCCACTAaACCCCATGCTTTCGGTTTTCCTCGACTCTCGAG ATACGGAGATCAGTT Trans-splicing have also been found in higher eukaryotes (Dorn, 2001) (www.wisc.edu/pharm)

24. Cis- vs. trans-splicing of pre-mRNAs Single RNA substrate Two RNA substrates (Blumenthal, WormBook)

25. Same splicing mechanism is employed in trans-splicing pre-mRNA splicing trans-mRNA splicing spliced leader Spliced leader contains the cap structure! (www.wisc.edu/pharm)

26. Alternative splicing: a way to increase total number of genes Alternative splicing: a single gene can encode many messages depending on how the message is spliced

27. Drosophila Dscam gene contains thousands of possible splice variants • Alternative possibilities for 4 exons leave a total number of possible mRNA variations at 38.016

28. Common forms of alternative splicing

29. CTD of RNA pol II plays important role in pre-mRNA splicing (Kornblihtt et al, 2004)

30. Effect of transcriptional elongation on alternative splicing (Kornblihtt et al, 2004) How do you experimentally test this?

31. Lecture 3 Summary