1. Chapter 15: Post-transcriptional events II: Capping and polyadenylation Cap structure

3. a. a phosphohydrolysis removes the terminal phosphate from a pre-mRNA; b. a guanylyl transferase adds the capping GMP. c and d. two methyl transferase methylate the N7 of the capping guanosine and the 2’ O-CH3 group of the penultimate nucleotide. Sequence of events in capping

4. Cap structure DEAE-cellulose chromatographic purification of vaccinia virus cap ,  - 32pGTP S-adenosyl[methyl- 3 H] methionine

5. G 

6. Identification of the capping substance as 7- methyl-guanosine. Miura and Furuichi ,  - 32 P-ATP S-adenosyl[methyl- 3 H] methionine  - 32 P-ATP  unable to be retained in the cap.  -phosphate was Alkaline phosphatase resistant  -phosphate was protected by substance X

7. Phosphodiesterase; phosphomonoesterase A Paper chromatography electrophoresis

8. Caps are made in steps : 1., a phosphohydrolysis removes the terminal phosphate from a pre- mRNA; 2. a guanylyl transferase adds the capping GMP. 3. two methyl transferase methylate the N7 of the capping guanosine and the 2’ O-CH3 group of the penultimate nucleotide. Sequence of events in capping

9. Identification of ppGpC as an intermediate in reovirus cap synthesis PPi electrophoresis Alkaline phosphatase, ppGpC  GpC Ion-exhanger column

10. Functions of Caps Effect of cap on RNA stability - protection Furuichi et al. Capped - m7GpppG (green) or blocked -GpppG (blue) glycerol gradient ultracentrifugation 8 h Wheat germ, 8 h Remove cap or block

11. Effect of cap on translatability D. Gallie; in vivo assay

12. Capping of U1 snRNA is necessary for its transport to the cytoplasm Hamm & Mattaj U1- RNA Pol II U6- RNA Pol III U1 driven by RNA Pol III U1-5: m2,2,7 G U6: no cap U1- m7G (nucleus)  Cytoplasm, receives other two methylation;complexed with proteins  nucleus to take part in RNA splicing Does the capping play role in transporting RNA out of the nucleus?

13. Mutant U1:unable to complex with proteins

14. (1) protection of the mRNA from degradation; (2) enhancement of the mRNA’s translatability; (3) transport of the mRNA out of the nucleus; (4) proper splicing of the pre-mRNA. Summary- the cap provides:

15. Polyadenylation Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nucleotides long at their 3’ends. This poly(A) is added post- transcriptionally by poly(A) polymerase.

16. Sheines & Darnell radioactively labeled HeLa cells for a short time (12 min); isolated hn RNA (nuclei) and mRNA (cytoplasm); RNase T1 (cut G), A (cut C or U)  (Ap)n

17. Finding poly(A) at the 3’end of hnRNA and mRNA

18. Effect of poly(A) on translation of globin mRNA in oocytes Revel et al. Globin mRNA(poly A + ) or (poly A - ) injected to frog oocytes; labeled Hb with 3 H-histidine; Sephdex G-100 column filtration

19. Effect of poly(A) on translation of globin mRNA in oocytes Revel et al. poly A + poly A -

20. Time course of translation of poly(A) + and poly(A) - globin mRNA. poly(A) + poly(A) -

21. Munroe and Jacobson Effect of poly (A) on translatability and stability of mRNAs

22. Effect of poly(A) on recruitment of mRNA to polysomes Munroe & Jacobson Poly(A) enhances lifetime and translatability. But, relative importance varies with system

23. (a) cutting, (b) polyadenylation, (c) degradation Basic Mechanism of Polyadenylation

24.  -Globin gene transcription extends beyond the poly(A) site. Hofer & Darnell Isolated nuclei from DMSO stimulated red blood cells; run-on transcription with 32 P-UTP; hybridized with DNA probes (A,B,….F) of  -Globin gene

25. Adenovirus late transcription unit Poly(A)

26. Model 3. Transcripts are clipped and polyadenylated while transcription is still in processs Nevins & Darnell Model 1. Stop at the coding region and polyadenylation Model 2. Stop at the very last end and polyadenylation

27. A B C D E DNA probes If model 1 is correct, then Chance of hybridization high low Not supported by experimental results

28. Nevins & Darnell

29. Basic Mechanism of Polyadenylation Where?

30. Summary of data on 369 veterbrate polyadenylation

31. Importance of the AATAAA sequence to polyadenylation Fitzgerald & Shenk Recombinant SV40 virus But, AATAAA is not sufficient. Deletion of immediate down stream region of the site can disrupt the polyadenylation

32. AATAAAA-N(23/24)-GT rich region-T rich region Gil and Proudfoot  -globin gene

34. Splicing happens before polyadenylation

35. Cleavage and poly-adenylation of a pre-mRNA A model for the pre-cleavage complex

36. Both PAP and CPSF are necessary for polyadenylation M. Wickens et al. Initiation of Polyadenylation

37. Polyadenylation has two phases Sheets & Wickens

38. CPSF binds to the AAUAAA motif Keller et al. 35 and 160 Kd proteins

39. Polyadenylation requires both cleavage of the pre-mRNA and polyadenylation at the cleavage site. Cleavage in mammals requires : CPSF, CstF, CF1 and CFII, and poly(A) polymerase (PAP). Polyadenylation has two phases. Once the poly(A) reaches about 10 nt in length, further polyadenylation becomes independent of the AAUAAA signal and depends on the poly (A) itself. Summary

40. Purification of poly(A)-binding protein (PABII) E. Wahle Elongation of Polyadenylation 49 Kd protein Activity assay Nuclear protein

41. Effect of CPSF and PABII on polyadenylation

42. Elongation of poly(A) requires PAB II. This protein binds to a pre- initiated oligo (A) and aids poly(A) polymerase in elongating poly(A) up to 250 nt. PAB II acts independently of the AAUAAA motif. It depends on poly(A), but its activity is enhanced by CPSF. PAP CPSF PABII CFI, II, CstF Summary

44. Architecture of PAP Specific polyadenylation carried out by full-length and C-terminally truncated PAP Manley et al.

45. Shortening of cytoplasmic poly(A) Sheines & Darnell Turnover of Poly(A) 48 h labeling Cytoplasmic poly(A) RNA Nuclear poly(A) RNA Summary - Poly(A) turns over in the cytoplasm. RNase tears it down, and PAP builds it back up. When the poly(A) is gone, the mRNA is slated for destruction. 12 min labeling

46. Dependence of PAN on PAB I, and distributive nature of PAN Sachs et al.

47. Biphasic de-adenylation Sachs et al.

48. Cytoplasmic deadenylation is carried out by PAN (poly(A) nuclease), in conjunction with PAB I (poly(A) binding protein). This reaction is biphasic. Rapid and slow phases (terminal 12-25 nt). Summary-

49. Various rates of de-adenylation in yeast mRNAs Sachs et al.

50. A sequence in mRNA 3’UTR that inhibits terminal deadenylation Summary of 3’UTR mutations and their effects on de-adenylation Sachs et al.

51. Deadenylation is not equally efficient for all mRNAs. The 3’UTR controls the efficiency of de-adenylation. An adenine-uridine-rich (ARE3) about 60 nt upstream of the poly(A) tail is a sensitive site. Summary-

52. Cytoplasmic poly-adenylation Maturation-specific poly-adenylation in frog oocytes; Maternal RNA[Poly(A) - ]; D7 RNA polyadenylated.

53. Cytoplasmic poly-adenylation Maturation-specific poly-adenylation of two RNAs Wickens et al.

54. UUUUUAU confers maturation specific poly-adenylation Abolition of maturation specific poly-adenylation by mutations in the AAUAAA motif

55. The Effect of the Cap and Poly(A) on Splicing (1) protection of the mRNA from degradation; (2) enhancement of the mRNA’s translatability; (3) transport of the mRNA out of the nucleus; (4) proper splicing of the pre-mRNA. The Cap function:

56. Shimura et al Production of capped and uncapped splicing substrates Effect of cap on splicing a substrates with two introns HeLa nuclear extract

57. Effect of CBC on splicing and pre-splicesome formation Mattaj et al Cap binding complex: CBP80 and CBP20 Activity assay Western blotting Splicesome complex assay

58. Summary- Removal of the first intron from model pre- mRNAs in vitro is dependent on the cap. This effect may be mediated by CBC that is involved in spliceosome formation.

59. Effect of polyadenylation on splicing a pre-mRNA with a single intron. Niwa & Berget

60. Effect of poly-adenylaton on splicing a two-intron substrates

61. Summary- polyadenylation of model substrates in vitro is required for active removal of the intron closest to the poly(A). However, splicing any other introns out of the these substrates occurs at a normal rate even without polyadenylation.