Elongation and pre-mRNA processing

1. Elongation and pre-mRNA processing

2. Introduction • Regulation of elongation • most transcription units are probably regulated during elongation because the elongation machinery must coordinate with so many other nuclear processes while navigating a nucleoprotein template. • Regulation can be general, applying to many genes, or selective. • Themes • RNA polymerase II • Elongation factors - Specific proteins affecting elongation • Chromatin and elongation • Pre-mRNA processing • Capping, Splicing and Termination/3´-end formation

3. The elongating RNAPII

4. Control of elongation by RNAPII • Two basic features • RNAPII has a remarkable processitivity • RNAPII is susceptible to transient pausing and arrest

5. The Elongation phase - RNAPII • Processivity explained by 3D of RNAPII • RNAP closes upon”promoter clearance” and transition to TEC (trx elongation complex) • Contacts to PIC disrupted and new contacts with elongation factors formed • CTD phosphorylated • Conformational change to a ternary complex of high stability • Closed chanel around DNA-RNA hybrid in the active site Encouter obstacles that lead to to pausing or arrest

6. Early evidence for elongation factors • Early evidence for general elongation factors • Elongation rate of RNAPII in vitro << in vivo • In vitro: 100-300 nt per min, frequent pauses, some times full arrest • In vivo: 1200-2000 nt per min, probably because elongation-factors suppress pausing • The DRB-inhibitor: nucleotide-analogue causing strong inhibition of hnRNA synthesis, acts by enhanced arrest of RNAPII, but has no effect on purified RNAPII, targets probably an elongation factor • DRB = 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole

7. Several protein-factors isolated that stimulate elongation P-TEFb TFIIS TFIIF Elongin FACT ELL

8. Stimulation of elongation - multiple mechanisms • Several possible mechanisms stimulating elongation • Factors that facilitate elongation through chromatin • FACT - ”facilitates chromatin transcription” • SWI/SNF-type chromatin remodelling • Factors that facilitate elongation by supression of RNAPII pausing • TFIIF, Elongin (SIII), ELL, ELL2, CSB • Factors that facilitate elongation by liberating RNAPII from arrest • TFIIS Elongation RNAPII target Chromatin target Suppress Pausing Increase rate Suppress arrest

9. Elongation factors that target RNAPII Phosphorylation of CTD - early stages of elongation

10. Elongation factors that target RNAPII: P-TEFb phosphorylates CTD • The P-TEFb (positive transcription elongation factor) complex, which contains the cyclin-dependent kinase CDK9 and cyclin T, couples RNA processing to transcription by phosphorylating Ser2 of the RNAP II CTD • Identified biochemically • Based on its ability to protect RNAPII aginst arrest in a Drosophila tr.system • structure: Heterodimer = 124 kDa + 43 kDa • activity: a CTD-kinase • Cdk9 (også kalt PITALRE) + cyclin T1, T2 or K • Kinase-inactive form without effect on elongation • Distinctive feature: inhibited by the nucleotide analogue DRB

11. Elongation - phosphorylation cycle

12. P-TEFb action - release from DSIF/NELF induced pause • Mechanism of action • Ser 2 phosphorylation of CTD with P-TEFb blocks binding of the elongation -inhibitors NELF and DSIF (DRB-sensitivity inducing factor) • NELF = Negative ELongation Factor, a multiprotein complex (5 polypeptides 46-66 kDa) • DSIF = DRB Sensitivity Inducing Factor, a heterodimer (14 + 160 kDa) = Spt4 + Spt5 in yeast • Stop and wait for capping: DSIF interacts with hypophosphorylated CTD. NELF recognizes the RNAP II–DSIF complex and halts elongation. This pause allows the recruitment of the capping enzyme by the CTD and DSIF (Spt5 subunit), which adds a 5 -cap to the nascent transcript.

13. The first steps in elongation • The kinase action of TFIIH phosphorylates Ser5 of CTD • DSIF and NELF followed by the capping machinery (CE) are recruited into the stalled transcription complex • CE caps the nascent mRNA (see later) • SCPs (small CTD phosphatases) dephosphorylate Ser5. • P-TEFb phosphorylates Ser2 of CTD + the SPT5 subunit of DSIF, which may facilitate the release NELF. • Trx elongation is resumed through the association of elongation factors (EFs).

14. HIV and P-TEFb P-TEFb Cdk9 Cyclin T Human specific Tat 5´- TAR CTD-kinase CTD RNAPII Stimulated elongation

15. HIV and P-TEFb • HIV-1 produces its own”elongation factor” Tat • Tat is a sequence-specific RNA-binding protein encoded by HIV • Tat binds to a sequence-element TAR (transactivation response element) in the 5´-end of HIV-transcripts • Tat+TAR promote effective elongation of HIV transcripts • P-TEFb + CTD is required for Tat-function • Ternary complex formed with human cyclin T1+ Tat + TAR (human, not murine T1) • Murine cells become HIV-infectable after transfection with human cyclin T1 • Mechanism: Tat facilitates HIV expression by recruiting P-TEFb to TAR, which improves specifically elongation of HIV-transcripts

16. Elongation factors Helping arrested RNAPII

17. Pausing and arrest • RNAPII encounters obstacles during elongation leading to pausing or arrest • This stage of trx is subject to control and several genes may be regulated also on the level of elongation • Pausing and arrest • result from aberrant backward movement of RNAPII, leading to displacement of the 3´-OH end of the growing RNA from the catalytic site • Pausing = reversible • Arrest = not reversible

18. Elongation factors that suppress RNAPIIarrest : TFIIS • structure: monomer = 38 kDa • TFIIS binds arrested RNAPII and • TFIIS strongly enhances a weak intrinsic nuclease activity of RNAPII • TFIIS induces the polymerase to cleave its nascent transcript, repositioning the new RNA 3´-end within the polymerase catalytic center

19. TFIIS helps RNAPII to recover from an arrested state & resume elongation • Arrest and resuce • When RNAPII approaches an arrest site, it may stop, reverse direction (backtracking), and extrude RNA, leading to transcriptional arrest. • TFIIS can rescue arrested Pol II by inducing cleavage of the extruded RNA fragment. Transcription is then resumed and continued past the arrest site.

20. RNAPII active site switches from polymerizing to cleavage • RNAPII contains a single active site for both RNA polymerization and cleavage polymerizing TFIIS induced cleavage

21. Elongation factors Helping paused RNAPII

22. Pausing and arrest • Pausing = rate-limiting step during elongation • RNAPII susceptible to pausing at each step • RNAPII cycles between active and inactive (paused) conformations

23. Elongation factors affecting pausing or arrest

24. Elongation factors that suppresspausing: TFIIF, Elongin (SIII), and ELL • TFIIF • Protects the elongation complex against pausing • Acts probably by a direct but transient interaction with the elongating RNAPII • phosphorylation of RAP74 stabilizes binding to RNAPII and stimulates elongation

25. Elongation factors that suppresspausing: TFIIF, Elongin (SIII), and ELL • Elongin • Heterotrimer of subunits A, B and C where A is active, B and C regulatory • Elongins activity can probably be regulated by the von-Hippel-Lindau (VHL) tumor supressor protein which binds Elongin BC and blocks their binding to Elongin A. • Genetic disease VHL dispose for several cancers, where mutated VHL binds Elongin BC less avidly • ELL • 80 kDa elongation factor found fused with MLL (mixed lineage leukemia) in certain leukemias with translocation between chromosome 11 and 19 (ELeven-nineteen Leukemia)

26. Mechanims of action • Pausing = rate-limiting step during elongation • RNAPII susceptible to pausing at each step • RNAPII cycles between active and inactive (paused) conformations • Elongation factors that suppress pausing, probably act by decreasing the fraction of time RNAPII spends in an inactive paused conformation • For many factors supressing pausing and increasing rate of trx, our understanding of mechanism is incomplete

27. Elongation factors helping RNAPII through chromatin

28. Elongation through chromatin • Chromatin is not only an obstacle to TFIID binding and PIC assembly, but also for the elongating RNAPII

29. Through arrays of nucleosomes - propagation of chromatin disruption • Nucleosome arrays more difficult to pass • Inter-nucleosome contacts repress elongation • Induce pausing • Some elongation factors stimulate elongation on free DNA in vitro, but cannot overcome the chromatin block

30. Through arrays of nucleosomes - propagation of chromatin disruption • In vivo cellular factors helps to disrupt the chromatin block to elongation

31. Elongation factors acting on chromatin • Factors that facilitate elongation through chromatin • SWI/SNF-type chromatin remodellering through ATPdependent mechanisms • Swi-Snf and Chd1 remodel nucleosomes • Proteins that acetylate (e.g. Gcn5 and Elp3) or methylate histones • FACT - ”facilitates chromatin transcription”- can bind to and destabilize nucleosomes • a heterodimer where SPT16 encodes the large subunit • HMG1-like factor SSRP1 • Proposed that FACT transiently binds and removes H2A+H2B • Spt4+Spt5 (DSIF) and SPT6 proteins • Reassembly of chromatin after passage of RNAPII important • To suppress trx initiation from cryptic initiation site (noise) • FACT and SPT6 probably acts by enabling chromatin structure to be disrupted and then reestablished during trx

32. The targeting problem again • How are these factors targeted to the transcribed regions of the genome? • Hitching a ride on the RNAPII • Likely through recognizing hyperphosphorylated CTD • P/CAF (HAT) binds specifically to the hyperphosphorylated RNAPII • An ”elongator” isolated in yeast that associates only with the hyperphosphorylated elongating form of RNAPII • One of the subunits, Elp3 = HAT

33. FACT facilitates chromatin transcription • FACT is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. • FACT specifically interacts with nucleosomes and histone H2A/H2B dimers • FACT appears to act as a histone chaperone to promote H2A/H2B dimer dissociation from the nucleosome and allow RNAPII transcription on chromatin • Trx correlates with the generation of a nucleosome depleted for one H2A/H2B dimer

34. FACT • FACT functions to destabilize the nucleosome by selectively removing one H2A/H2B dimer, thereby allowing RNAP II to traverse a nucleosome.

35. The ebb and flow of histones • The histone chaperone activity of Spt6 helps to redeposit histones on the DNA, • thus resetting chromatin structure after passage of the large RNAPII complex. • FACT enables the displacement of the H2A/H2B dimer from the nucleosome, leaving a “hexasome” on the DNA. • The histone chaperone activity of FACT might help to redeposit the dimer after passage of RNAPII, thus resetting chromatin structure. • A possible relationship between histone acetylation and transcription through the nucleosome. • In this scenario, HATs associated with RNAPII acetylate the histone that is being traversed, facilitating its disruption and displacement. • Upon redeposition of the displaced histone dimer or octamer, HDACs immediately deacetylate the histones, resetting chromatin structure.

36. Three possible disruption mechanisms • 1. A decompaction of chromatin surrounding the activator site • Implies a specialized ”pioneer” polymerase to do the first trip • Implies that elongation itself could play a role in chromatin modification • 2. Activators promote decompaction of chromatin over the whole gene • 3. Histone methylation

37. Histone Lys methylation • PIC assembly • Upstream and downstream of the PIC, nucleosomes are dimethylated on H3-K4 and not methylated at H3-K36. • Promoter clearance • CTD-kinase of TFIIH phosphorylates ser-5 of the CTD resulting in disengagement from the promoter and recruitment of the Set1 complex (HKMT) and the capping machinery. • Elongation • CTK1 kinase complex (or P-TEFb) is recruited to the trx apparatus resulting in phosphorylation of ser-2 of the CTD. Ser-5 Ser-2

38. HKMT (SET1) HKMT (SET2) Histone methylation = stable epi-mark RNAPII dynamic process

39. In yeast two separate HKMT-containing complexes associate with RNAP II and are implicated in histone methylation at mRNA coding regions • Set1 is implicated in establishing H3-K4 histone methylation. • Set2 is implicated in establishing H3-K36 histone methylation. • tri-methylation of H3-K4 catalyzed by Set1 accumulate near the 5´-mRNA coding region of genes • and is associated with the early stages of transcription. • Set2 specifically associates with the elongating form of RNAP II. • Set2-mediated H3-K36 methylation, along with di-methyl H3-K4, corresponds to later stages of elongation.

40. PAF complex • The yeast Set1 and Set2 HKMTs are recruited by the PAF trx elongation complex in a manner dependent upon the phosphorylation state of the CTD of RNAPII • The PAF complex directly recruits Set1 to the trx machinery by bridging the interaction between RNAP II and Set1 • PAF has five subunits • Paf1, Rtf1, Cdc73, Leo1, and Ctr9 • Evidence suggests that PAF integrates transcriptional regulatory signals and coordinates modifications affecting chromatin

41. A possible logic? • The CTD of RNAPII has been found to anchor several proteins with a role in elongation and pre-mRNA processing • A histone code of methylated histone-tails may provide additional anchorage sites for elongation factors or processing enzymes Ass factors Ass factors

42. Pre-mRNA processing Processes tightly linked to elongation

43. Pre-mRNA (hnRNA) AAAAAAAAAAAAA cap mRNA A role for CTD in mRNA processing? • Several novel CTD-binding proteins identified the last few years with functions in splicing and termination • Tight coupling : transcription - pre-mRNA processing

44. CTD-mediated coupling : transcription - pre-mRNA processing • Pre-mRNA processing • Capping • Splicing • Cleavage/polyadenylation • Physical contact between the machines for for transcription and pre-mRNA processing through CTD

45. Capping

46. Cotranscriptional ”Capping” • Pre-mRNA modified with 7-methyl-guanosine triphosphate (cap) when RNA is only 25-30 bases long • Cap: 3 modifications • 7-met-guanosine coupled to 5´-end • Coupling by 5´-5´triphosphate bridge • Takes place co-transcriptionally • O2´-methylation of ribose • Cap2, Cap1 (multicellulær), Cap0 (unicellulær) • N6-methylation of adenine • Capping occurs co-transcriptionally Cap-1 Cap-2

47. Capping • 3 enzymes involved • 1. RNA 5´-Triphosphatase (RTP) removes a phosphate • 2. Guanylyl transferase (GT) attach GMP • Enzyme 1+2 coupled: in multicellular organisms: in same polypeptid, in yeast heterodimers • 3. 7-methyltransferase (MT) modifies the terminal guanosine 2. 1. 3.

48. Cotranscriptional ”Capping” • CTD recruits capping enzyme as soon as it is phosphorylated • CTD required for effective capping • Guanylyl transferase (mammalian + yeast) binds directly to phosphorylated CTD, not to non-phosphorylated • 7-methyltransferase (yeast) binds also phosphorylated CTD • phosphorylated CTD may also regulate the activity of the enzymes • Cap structure is recognized by CBC (Cap binding complex) • Composed of two proteins CBP20 and CBP80 • Major role in stabilization, block exonucleases • CBC stimulates subsequent splicing and 3´-end processing

49. Splicing

50. Splicing • Splicing of introns occurs cotranscriptionally • EM evidence • Half-life BR1 intron only 2.5 min ≈ 5 kbs elongation of RNAPII • Splicing depends on CTD • Inhibited by CTD truncation • In vitro splicing stimulated by added phosphorylated CTD • CTD binds probably splicing-factors • Not fully characterized • CTD associated with SR- and Sm-splicing factors