pre-mRNA processing and decay
- Created by: natasha8sherry
- Created on: 14-01-14 11:47
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- pre-mRNA processing and decay
- several steps of processing that help to protect the mRNP particle from premature degradation in the cytoplasm
- addition of 5'cap
- modified guanosine base bound to the final residue through a 5'-5' triphosphate linkage
- phosphatase removes the terminal phosphate
- a guanylytransferase adds the gtp
- a methyltransferase methylates the 7-position to create the 7-methylguanosine cap structure
- these enzymes are all part of the capping enzyme complex which is bound to RNAP II before initiation of transcription, ensuring the 5' cap is added to all mRNAs
- cap protects the 5' end from exonucleolytic degradation by Xrn1 (as it cannot recognise the guanosine)
- another enzyme complex - the cap-binding complex, binds to the 5' cap as it is added, further protecting the mRNA molecule by preventing decapping enzymes from removing the cap
- Polyadenylation
- coupled with 3' cleavage, polyadenylation is stimulated by the cleavage reaction
- polyadenylate polymerase begins to add a tail of AMP molecules to the 3' end
- then recruitment of PAB2 which promotes faster polyadenylation by PAP
- protects the 3' end from degradation by exosome (as well as promoting nuclear export)
- other not-as-important processes
- splicing - removal of non-coding introns
- leads to recruitment of several proteins that remain bound to the transcript until it is exported
- these proteins ensure the full and proper mRNP is assembled, protecting it from endonucleolytic cleavage
- leads to recruitment of several proteins that remain bound to the transcript until it is exported
- splicing - removal of non-coding introns
- modified guanosine base bound to the final residue through a 5'-5' triphosphate linkage
- nonsense-mediated decay
- used when we see NONSENSE MUTATIONS that cause a premature stop codon
- this causes the synthesis of non-functional and deleterious truncated proteins
- now being used as therapeutic drug targets using compounds which makes the translation mechanism bypass stop codons
- process that eliminates mRNAs carrying premature stop codons in euks
- requires 2 ingredients
- ribosome stalled at a stop codon
- downstream cis-acting sequence
- the codon itself is not important, but the stalled ribosome is, that is somehow sensed by the nonsense-mediated decay (NMD) machinery
- NMD works in a broader sense of mRNA surveillance
- targets the products of aberrant splicing (when an intron is not correctly spliced out). and is likely to introduce a stop codon which needs to be eliminated
- targets non-functional ncRNAs that are expressed inappropriately by transposons or teroviral sequences in the genome
- NMD works in a broader sense of mRNA surveillance
- e.g. in yeast
- cis-acting sequence is downstream sequence element from premature termination codon (PTC) or the 3' UTR itself
- initiated by decapping, followed by 5'-3' decay mediated by Xrn1
- e.g. in mammals
- dependent on complex called exon junction complex, deposited approx 20 nucleotides upstream of exon-exon boundaries in splicing process
- exon junction complex has role in mRNP export, translation, mRNP cytoplasmic localisation and mRNA stability
- composed of 4 proteins
- RNA helicase (eIF4A-III)
- help to anchor the complex to appropriate site on mRNA
- requires surveillance complex which monitors mRNAs for inappropriate stop codons
- includes UPF proteins
- e.g. RNA helicase Upf1
- interacts with translation release factors eRF1 and eRF3
- recruited by eRF1 and eRF3 after the ribosome encounters a stop codon
- then interacts and recruits factors involved both in 5'-3' decay and 3'-5' decay
- e.g. RNA helicase Upf1
- includes UPF proteins
- after the 1st pioneer round of translation, exon junction complexes are removed, also removing Upf2 and Upf3 in the process
- NMD therefore does not occur in pioneer translation because the UPF proteins are unable to recruit mRNA degradation factors
- however, when translation stalls due to a premature termination codon, Upf1 is recruited and there is time for the surveillance complex with Upf2 and Upf3 --> mRNA degredation
- dependent on complex called exon junction complex, deposited approx 20 nucleotides upstream of exon-exon boundaries in splicing process
- preventing nonsense-mediated decay at the true stop codon
- exon-exon boundaries are generally not juxtaposed to true stop codons
- possibly other forms of premature stop codon recognition which also exist in eukaryotes and that NMD may not be totally dependent on the exon junction complex system
- Mex67-5 blocks nonsense-mediated decay
- Mex67 is major RNA export receptor (TAP in mammals), but specific mutants deficient in part of the Mex67 protein (Mex67-5 mutant) have blocked NMD
- used when we see NONSENSE MUTATIONS that cause a premature stop codon
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