pre-mRNA processing and decay

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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
    • 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
      • 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
        • 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
      • 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

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