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Chapter 15 : Post-transcriptional events II: Capping and polyadenylation

Chapter 15 : Post-transcriptional events II: Capping and polyadenylation. Cap structure. Sequence of events in capping. a. a phosphohydrolysis removes the terminal phosphate from a pre-mRNA; b . a guanylyl transferase adds the capping GMP.

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Chapter 15 : Post-transcriptional events II: Capping and polyadenylation

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

  2. Sequence of events in capping • 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.

  3. Cap structure • DEAE-cellulose chromatographic purification of vaccinia virus cap b, g- 32pGTP S-adenosyl[methyl-3H] methionine

  4. a-b-a G

  5. Identification of the capping substance as 7-methyl-guanosine. • Miura and Furuichi b, g- 32P-ATP S-adenosyl[methyl-3H] methionine g- 32P-ATPunable to be retained in the cap. b-phosphate was Alkaline phosphatase resistant b-phosphate was protected by substance X

  6. Phosphodiesterase; phosphomonoesterase electrophoresis A Paper chromatography

  7. Sequence of events in capping • 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.

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

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

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

  11. Capping of U1 snRNA is necessary for its transport to the cytoplasm Hamm & Mattaj 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? U1- RNA Pol II U6- RNA Pol III U1 driven by RNA Pol III

  12. Mutant U1:unable to complex with proteins

  13. Summary- the cap provides: • (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.

  14. 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.

  15. 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

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

  17. 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 3H-histidine; Sephdex G-100 column filtration

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

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

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

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

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

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

  24. Adenovirus late transcription unit Poly(A) Poly(A)

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

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

  27. Nevins & Darnell

  28. Basic Mechanism of Polyadenylation Where?

  29. Summary of data on 369 veterbrate polyadenylation

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

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

  32. Splicing happens before polyadenylation

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

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

  35. Sheets & Wickens Polyadenylation has two phases

  36. 35 and 160 Kd proteins Keller et al. CPSF binds to the AAUAAA motif

  37. Summary • 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.

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

  39. Effect of CPSF and PABII on polyadenylation

  40. Summary CFI, II, CstF • 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. CPSF PAP PABII

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

  42. Turnover of Poly(A) Sheines & Darnell Nuclear poly(A) RNA Cytoplasmic poly(A) RNA 48 h labeling 12 min labeling Shortening of cytoplasmic poly(A) • 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.

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

  44. Sachs et al. Biphasic de-adenylation

  45. Summary- • 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).

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

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

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