Diversité des transcrits: Les sites de polyadenylation « intergéniques »
Chapter 15 : Post-transcriptional events II: Capping and polyadenylation
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Transcript of Chapter 15 : Post-transcriptional events II: Capping and polyadenylation
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Chapter 15: Post-transcriptional events II: Capping and polyadenylation
• Cap structure
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• 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
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• Cap structure
• DEAE-cellulose chromatographic purification of vaccinia virus cap
, - 32pGTPS-adenosyl[methyl-3H] methionine
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G
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• Identification of the capping substance as 7-methyl-guanosine.
• Miura and Furuichi
, - 32P-ATPS-adenosyl[methyl-3H] methionine
- 32P-ATPunable to be retained in the cap.-phosphate was Alkaline phosphatase resistant-phosphate was protected by substance X
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Phosphodiesterase; phosphomonoesterase
A
Paper chromatography
electrophoresis
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• 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
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Identification of ppGpC as an intermediate in reovirus cap synthesis
PPi
electrophoresis
Alkaline phosphatase, ppGpC GpC
Ion-exhanger column
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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
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Effect of cap on translatabilityD. Gallie; in vivo assay
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Capping of U1 snRNA is necessary for its transport to the cytoplasmHamm & Mattaj
U1- RNA Pol IIU6- RNA Pol III
U1 driven by RNA Pol III
U1-5: m2,2,7 GU6: no cap
U1- m7G (nucleus) Cytoplasm, receives other two methylation;complexed with proteins nucleus to take part in RNA splicingDoes the capping play role in transporting RNA out of the nucleus?
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Mutant U1:unable to complex with proteins
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• (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:
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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.
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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
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Finding poly(A) at the 3’end of hnRNA and mRNA
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Effect of poly(A) on translation of globin mRNA in oocytesRevel et al.
Globin mRNA(poly A+) or (poly A-) injected to frog oocytes; labeled Hb with 3H-histidine; Sephdex G-100 column filtration
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Effect of poly(A) on translation of globin mRNA in oocytesRevel et al.
poly A+
poly A-
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• Time course of translation of poly(A)+ and poly(A)- globin mRNA.
poly(A)+
poly(A)-
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Munroe and Jacobson
Effect of poly (A) on translatability and stability of mRNAs
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Effect of poly(A) on recruitment of mRNA to polysomes
Munroe & Jacobson
Poly(A) enhances lifetime and translatability. But, relative importance varies with system
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• (a) cutting ,
• (b) polyadenylation,
• (c) degradation
Basic Mechanism of Polyadenylation
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-Globin gene transcription extends beyond the poly(A) site.
Hofer & Darnell
Isolated nuclei from DMSO stimulated red blood cells; run-on transcription with 32P-UTP; hybridized with DNA probes (A,B,….F) of -Globin gene
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Adenovirus late transcription unit
Poly(A) Poly(A)
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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
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AB
CD
E
DNA probes
If model 1 is correct, then
Chance of hybridization high low
Not supported by experimental results
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Nevins & Darnell
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Basic Mechanism of Polyadenylation
Where?
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Summary of data on 369 veterbrate polyadenylation
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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
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AATAAAA-N(23/24)-GT rich region-T rich region
Gil and Proudfoot
-globin gene
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Splicing happens before polyadenylation
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Cleavage and poly-adenylation of a pre-mRNA
A model for the pre-cleavage complex
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Both PAP and CPSF are necessary for polyadenylation
M. Wickens et al.
Initiation of Polyadenylation
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Polyadenylation has two phases Sheets & Wickens
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CPSF binds to the AAUAAA motif
Keller et al.
35 and 160 Kd proteins
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• 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
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Purification of poly(A)-binding protein (PABII)E. Wahle
Elongation of Polyadenylation
49 Kd proteinActivity assay
Nuclear protein
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Effect of CPSF and PABII on polyadenylation
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• 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.
PAPCPSF
PABII
CFI, II, CstF Summary
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Architecture of PAP
Specific polyadenylation carried out by full-length andC-terminally truncated PAP
Manley et al.
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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
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Dependence of PAN on PAB I,and distributive nature of PAN
Sachs et al.
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Biphasic de-adenylation
Sachs et al.
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• 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-
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Various rates of de-adenylation in yeast mRNAs
Sachs et al.
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A sequence in mRNA 3’UTR that inhibitsterminal deadenylation
Summary of 3’UTR mutations and theireffects on de-adenylation
Sachs et al.
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• Deadenylation is not equally efficient for all mRNAs.• The 3’UTR controls the efficiency of de-adenylation. An adenin
e-uridine-rich (ARE3) about 60 nt upstream of the poly(A) tail is a sensitive site.
Summary-
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Cytoplasmic poly-adenylation
Maturation-specific poly-adenylation in frog oocytes; Maternal RNA[Poly(A)-]; D7 RNA polyadenylated.
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Cytoplasmic poly-adenylation
Maturation-specific poly-adenylation of two RNAs
Wickens et al.
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UUUUUAU confers maturation specific poly-adenylation
Abolition of maturation specificpoly-adenylation by mutations in the AAUAAA motif
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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:
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Shimura et al
Production of capped and uncapped splicing substrates
Effect of cap on splicing a substrates with two introns
HeLa nuclear extract
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Effect of CBC on splicing and pre-splicesome formation
Mattaj et alCap binding complex: CBP80 and CBP20
Activity assayWestern blotting
Splicesome complex assay
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• 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.
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Effect of polyadenylation on splicing a pre-mRNA with a single intron.Niwa & Berget
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Niwa & Berget
Effect of poly-adenylaton on splicing a two-intron substrates
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• 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.