BIO 344 1st Edition Lecture 21Outline of Last Lecture I. Prionsa. Diseasesb. Structurec. transmissionOutline of Current Lecture I. mRNA degradationa. 5’ capb. Premature termination codonc. Exon-exon junctionII. Translation and degradation are opposing pathwaysa. Xrn1III. NMDa. EJCIV. Treating Disease due to PTCsCurrent LecturemRNA degradation- mRNA is unstable - factors that affect stabilityo 5’ cap Protects against 5’ exonuclease, rectuits eIF4Eo Premature termination codon (PTC) Targets transcript for nonsense-mediated decay (NMD)o Exon-exon junction Binding site for destabilizing or stabilizing proteinso AU-rich element (ARE)These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Translation and Degradation are Opposing Pathways- Factors that promote efficient translation are stable- Degradation required for remodeling for translation- General degradationo polyA tail gets shorter and shorter= degradation o when short enough, RNA susceptible to decapping by decapping enzyme Dcp2 then destroyed by Xrn1 exonuclease Xrn1 starts on 5’ end but does not work until the cap is removed- Needs 5’ phosphate Or exosome can degrade from 3’ end when polyA is removed- Exosome= RNA gets unwound, sent through channel, degraded, nucleotides come out- polyA binding protein (PAB) binds polyA and eIF4G to ensure tethering to RNA- PAB and degradation from 3’ endo RNA with polyA, add in deadenylase With no PAB, loses polyA tail in 5 minutes- So fast, we don’t see progression With PAB, polyA decreases gradually- Blocks progression of deadenylase- Happens more gradually slowing deadenylase- Continual competition between PAB and deadenylase Once polyA is too short, PAB cannot bind and EIF4G affinity for cap is also weakened - siRNA direct cleavage of mRNAo Xrn1 degrades 3’ fragmento Exosome degrades 5’ fragmentNMD- Quality control- Beta-thal due to mutations that introduce PTCs- Beta-globin transcripts are very stable—need to make tons of beta-globin/hemoglobin- Actinomycin D- inhibits transcription- When transcription stops, no mRNA accumulation—its being destroyed- An RNA with stop codon in premature position unstable nonsense codon NMD- PTC position is importanto Further away from normal stop position, short half life (more degradation)o Critical determinant distance between PTC and exon-exon junction (ligated post-splicing)o If PTC is at less than 50-55 nucleotides upstream from junction, will be recognized as premature and trigger NMD- Normal UAG downstream from junctiono Stop codon in last econ (95% of time)o Other 5% are in last< 50-55 nucleotide upstream of exon-exon junction- At end of splicingo Normal: after spliceosome leaves, exon junction complex (EJC) is left EJC sits 20-24 nucleotides upstream of exon-exon junction Translation starts and bumps of EJCo If PTC: first EJC bumped off, ribosome sees PTC and if still another EJC in front, targeted for NMD- Upf1 interaction with EJC triggers decappingo Driver of NMDo Recruited by termination factoro Sits longer on PTC than normal and binds to proteins of EJC, activating Upf1 (decapping) and degradationTreating Disease due to PTC’s- Can we pass PTC and utilize normal stop to translate full mRNA- Assay using luciferase—full length mRNA makes light- If we suppress termination, we suppress NMD because both EJCs get knocked off- UGA, UAG, UAA and gentomycino Gentomycin works on extracts but not whole cells because membrane is gone can get to ribosomeo PTC124 boosts luciferase expression in whole cells and extracts Drug that allows PTC to bypass Assay read through with Western blot Still not very efficient If it were 100% efficient, increase likelihood of ribosome adding an amino acid in place of PTC that causes protein to be nonfunctional—not frequent but possible Or might bypass both stops and make nonfunctional
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