28 - RNA Processing; Splicing

28 - RNA Processing; Splicing - mRNA processing...

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1 mRNA processing •Transcription and translation do not occur simultaneously in Eukaryotes •Eukaryote mRNAs (pre- mRNAs) require additional processing before translation: 5’ Capping (CTD tail) RNA splicing (CTD tail) Polyadenylation (CTD tail)
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2 mRNA processing: 5’ Capping Soon after RNAPII starts making transcript (chain size 20-30 nucleotides)- co- transcriptional modification(s) 5’G 1st base in original transcript (Lewin, 2006. Fig.7.18) 5’ 3 possible modifications •7-met-guanosine coupled to 5 ´ - end; binding through 5 ´ - 5 ´ triphosphate bridge (this always happens) •Methylation of ribose in Cap1 and Cap2 •N 6 -methylation of adenine possible in Cap1 and Cap2 (if adenine in first two positions)
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3 3 enzymatic reactions: 1. RNA 5 ´ -Triphosphatase (RTP) removes the 5’ phosphate 2. Guanylyl transferase (GT) attaches GMP 1 and 2 are parts of the same protein in higher eukaryotes 3. 7-methyltransferase (MT) modifies the terminal guanosine 1. 2. 3. Phosphorylated CTD recruits capping enzymes (they bind directly to phosphorylated CTD) Remember: function of 5’methyl cap - protection from degradation but also important role in splicing , nuclear transport and translation as well From: Cell, Vol. 108, 501–512, February 22, 2002 Integrating mRNA Processing with Transcription Nick J. Proudfoot, Andre Furger and Michael J. Dye 7-met-guanosine coupled to 5 ´ -end:
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4 mRNA processing: Polyadenylation 3 important elements in 3’UTR: 1. Polyadenylation signal = polyadenylation consensus element (except in yeast) 5’-AAUAAA-3’ Alternative: 5’-AUUAAA-3’ 2. Followed by 5’-YA-3’ (Y=pyrimidine, mostly C) in the next 11-20 nucleotides – cleavage and poly(A) addition site 3. Followed by GU-rich sequence Poly(A) is thought to help stabilization - binding site for cytoplasmic poly(A)- binding protein I ( PAB I ) to resist 3’-exonuclease action Role in splicing Increase of translation efficiency : rapid recycling of ribosomal subunits - function of citoplasmic PAB I - circularization of eukaryotic mRNAs Weaver, 3 rd edition
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3’ UTR: genomic and mRNA sequence e.g. cytoplasmic adenylation element, T/U-rich; for polyadenylation of maternal messages G-T rich involved in polyadenylation remember: those sequences “stay” in mRNA
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6 Polyadenylation cont. CPSF - cleavage and polyadenylation specificity factor – binds to AAUAAA CStF – cleavage stimulatory factor- binds to G/U region CFI & II – cleavage factor I & II-bending (not shown) ; mRNA is cleaved PAP - poly (A) polymerase binds Following cleavage: 1. slow phase (first 12 nucleotides) 2. rapid phase - requires nuclear poly (A) binding protein II ( PABPII )- 200- 250 nucleotides Role of CTD tail: to recruit enzymes necessary for polymerization
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7 Learning Objectives • Describe the mechanism of RNA splicing including the role of RNA-RNA and RNA- protein interactions • Define trans-splicing and its occurrence in different organisms Splicing of primary transcripts RNA processing & post-transcriptional control after transcription by RNAPII
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This note was uploaded on 12/23/2011 for the course BIOL 308 taught by Professor Miskovic during the Fall '09 term at Waterloo.

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28 - RNA Processing; Splicing - mRNA processing...

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