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15 Cards in this Set
- Front
- Back
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RNA processing pre-mRNA |
1. 5' Capping 2. Splicing and alternative splicing (can occur at the same time of polyA) 3. Poly A tail -length of poly A tail correlates with stablity mRNA 4. Quality control (incorrectly transcribed mRNA gets cleaved by endonuclease) |
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Cytoplasmic regulation of mRNA |
1. RNA stability -cytoplasmic polyA adenylation 2. RNA localization (neurons) -axons and dendrites that are far from nucleus mRNA needs to be transported 3. Translational control (general and specifc) |
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mRNA complex |
mRNA is always in a complex(never alone) with RNA binding proteins (ribonucleoproteins, mRNPs) |
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pre-mRNA processing coupling to transcription |
RNA pol II has C-terminal domain(regulated by phosphorylation) that confers specificity: -Many RNA processing factors are recruited by CTD result in local increase of the concentration.
YSPTSPS |
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CTD tail phosphorylation |
1. initially near TS start site Ser 5 phosphorylated 2. middle Ser2-P with Ser5-P 3. End Ser2-P -Ser5 dephosphorylated -prolines get isomerized --> cis become trans -trans proline recognized by RNA 3' end processing factors |
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5' end capping |
Protect mRNA from degradation cap consists of: 7-methylguanosine -phosphodiester bond 5' C of guanine links to 5'C of first nucleotide. 3 types of enzymatic activities: gamma-beta-alpha phosphate groups on the first nucleotide 1. phosphohydrolase: -cleaves gamma phosphate off
2. Guanyl transferase: -add GTP to beta-alpha P, alpha of GTP kept --> alpha(GTP)-beta-alpha P
3. Methyl transferase: -add methyl to 7th position of GTP and to first nucleotide
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Steps of 5'end capping |
1.PH gamma-alpha 2.GT alpha(GTP)-gamma-alpha 3.MT m-alpha(GTP)-gamma-alpha m-alpha(GTP)-gamma-alpha-nucleotide-m |
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eIF4E and cap protein |
In cytosol, cap protein serves as docking site for many molecules. eIF4e elongation factor(Translation) binds 5'Cap |
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PolyA adenylation |
2 poly A signals: AAUAAA is important sequence, 10-35nts upstream of polyA. G/U rich or U rich sequences 50 nt downstream polyA site |
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Cleavage vs polyA termination |
-mRNA transcribed beyond polyA site then cleavage occurs, not stop at polyA site. determined by: Pulse labeling viral primary transcripts showed RNA being transcribed beyond the site of polyadenylation. Then, in vitro reconstitution of RNA processing confirms |
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PolyA enzymess |
CPSF: Cleavage and polyadenylation specificity factor -endonuclease activity, won't cleave once PAP comes in --> 1 mechanism to ensure that the mRNA is not cleaved too early w/out polyA tail(3'end would be exposed). CStF: Cleavage stimulating factor CF: Cleavage factor PAP: Poly(A) polymerase PABPII: PolyA binding protein II |
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Steps of polyA |
1. CPSF recognizes/binds AAUAAA 2. CStF recognizes/binds G/U 3. CFI+CFII binds to CStF 4. PAP binds and polymerize a short stretch of poly A slowly 5. PABPII binds short stretch, stimulates PAP polymerizing (up to 200nt of polyA |
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Alternative(2) poly adenylation at 3'end |
1. CR-APA -alternate last exon, different poly A site -Two different proteins are synthesized(different C terminal) 2. UTR-APA -Multiple poly A sites -Result in different 3' UTR sequences |
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Alternative polyA of Ig heavy chain |
CR-APA heavy M chain 1. Resting B cell (membrane bound) -M1, M2 last exons(encodes transmembrane domain encoded) as alternate polyA site. 2. Activated B cell -first poly A site cuts off transmembrane domain coding exons.
First poly A site is weak -activated by an increase of CStF concentration. |
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Alternative polyA |
in cancer cells: -Often affects oncogenes 1.Cyclin D1/D2, FGF2, IMP-1 -3' UTR are shorter due to alternate polyA site(weaker and more upstream) -less suppression by miRNA silencing |
