mrna multiple processing and localization steps are required for proper mrna maturation following...
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![Page 1: MRNA Multiple processing and localization steps are required for proper mRNA maturation following the synthesis of the primary transcript. At a minimum,](https://reader036.vdocuments.mx/reader036/viewer/2022082411/56649d965503460f94a8003f/html5/thumbnails/1.jpg)
mRNA
• Multiple processing and localization steps are required for proper mRNA maturation following the synthesis of the primary transcript.
• At a minimum, these include capping, splicing, 3´-end cleavage, polyadenylation, and nuclear export.
• If each of these 5 processes are 90% efficient, only ~59% of mRNAs will be properly matured.
• What happens to the improperly processed transcripts? – Do they get sent to the cytoplasm for protein synthesis anyway?
– Do they get degraded before they can make potentially defective proteins?
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1. The splicing and processing components as well as export factors are co-transcriptionally recruited to the nascent transcript.2. The correct mRNA forms the core of an export-competent mRNP that is eventually exported and expressed in the cytoplasm.
Nuclear mRNA
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Post-transcriptional Processes II: Pre-mRNA Polyadenylation
• Most cytoplasmic mRNAs have a polyA tail (3’ end) of 50-250 Adenylates– a notable exception is histone mRNAs
• Added post-transcriptionally by an enzyme, polyA polymerase(s)
• Turns over (recycles) in cytoplasm
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Functions of the PolyA Tail
1. Promotes mRNA stability - Deadenylation (shortening of the polyA
tail) can trigger rapid degradation of the mRNA
2. Enhances translation- promotes recruitment by ribosomes- bound by a polyA-binding protein in the cytoplasm called PAB1- synergistic stimulation with Cap!
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Polyadenylation (PolyA) Signals
• AAUAAA in mammals and plants• Located ~20-30 bp from the polyA site
– Other hexamers less efficient but are used• Mutagenesis and in vivo expression studies
reveal 2 other motifs downstream of AAUAAA that are important:
1. GU-rich stretch2. U-rich stretch
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Polyadenylation (PolyA) Signals
• AAUAAA in mammals and plants• Located ~20-30 bp from the polyA site
– Other hexamers less efficient but are used• Mutagenesis and in vivo expression studies
reveal 2 other motifs downstream of AAUAAA that are important:
1. GU-rich stretch2. U-rich stretch
![Page 7: MRNA Multiple processing and localization steps are required for proper mRNA maturation following the synthesis of the primary transcript. At a minimum,](https://reader036.vdocuments.mx/reader036/viewer/2022082411/56649d965503460f94a8003f/html5/thumbnails/7.jpg)
Polyadenylation: Mechanism
• Occurs in 2 phases– Phase 1: requires AAUAAA and ~8 nt
downstream (3’) – Phase 2 : Once ~10 As are added, further
adenylation does not require the AAUAAA
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Proteins Required for Polyadenylation
Phase I:1. CPSF 2. PolyA polymerase
Phase II:3. PolyA polymerase (PAP II)4. PolyA Binding Protein II (PAB II)
- PAB II binds to short A-tail- Helps PAP synthesize long tails
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Cleavage and polyadenylation of pre-mRNAs in mammalian cells
1. Cleavage-and-polyadenylation specificity factor (CPSF) binds to an upstream AAUAAA polyadenylation signal.
2. CSTF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA; binding of CFI and CFII help stabilize the complex.
3. Binding of poly(A) polymerase (PAP) then stimulates cleavage at a poly(A) site, which usually is 10 – 35 nucleotides 3' of the upstream polyadenylation signal.
4. The cleavage factors are released, as is the downstream RNA cleavage product, which is rapidly degraded. Bound PAP then adds ≈12 A residues at a slow rate to the 3'-hydroxyl group generated by the cleavage reaction.
5. Binding of poly(A)-binding protein II (PABII) to the initial short poly(A) tail accelerates the rate of addition by PAP. After 200 – 250 A residues have been added, PABII signals PAP to stop polymerization.