BIO 344 1st Edition Lecture 19 Outline of Last Lecture I. RNAia. Introb. C. Elegansi. Par1ii. Sense and antisense with unc54II. Mechanism of RNAia. Dicerb. RISCi. siRNA and miRNAIII. Antiviral pathway in plantsa. GFP experimenti. System spread—amplificationIV. Human pathwaya. Apoptosisb. PKR triggering and preventionc. Genetic screen for host factors Outline of Current Lecture I. Micro RNAsa. Lin4 and lin14b. miRNA transcriptionc. miRNA silencingd. capCurrent LectureMicro RNA- miRNA= small RNas that silence certain cellular genes at the stage of protein synthesiso endogenous origino metazoanso 2,024 miRNAs in humans—more miRNA than transcription factorso Regulate ~60% protein-encoding genesThese 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.- C elegans—4 larval stageso Lin4—mutants stuck in a larval stage and never become adultso Lin14—mutants skip a larval stage and become adults precociouslyo When lin4 was cloned, mutation was found in a highly conserved region and caused a loss of function Turns out, this region is not a protein encoding region—discovered by identification of the 3’ splice site- Lin4 is an introno Lin4 sequence is complementary to lin14’s 3’ UTR Does the lin4 mutation affect lin14 expression?- Western blot (protein) and Northern blot (RNA) was performed and showed that when RNA was being made but not protein in mutantso Showed that is under translational controlo Lin4 binding to lin14 3’ UTR inhibits translation- Specific miRNAs control cell proliferation and apoptosis- miR-375 gene regulates insulin- mir genes control differentiation decisions for cell types- miRNA also play a role in development and maintenanceo development—AGO-/- is embryonic lethalo maintenance—antagomir (mir gene antagonist) binds and blocks miR-133 gene and causes enlarged heart in adults- miRNA transcriptiono expressed as long stem loopso miRNA cut from one side of loopo often folded into complex stem loop structure from which several miRNAs can beexpressedo RNAPII is utilizedo Pri-miRNA= processed pre mRNA by Drosha Leaves a 2 nucleotide 3’ overhang, like dicer Dicer recognizes and processes in cytoplasmo Bulges= nucleotides that aren’t base paired- How miRNAs silenceo RISC with guide RNA and Ago Different Ago proteins—- Ago2= slicer activity—mRNA cleaving- Ago1,3,4= work with miRNA to inhibit translationo Bulge distorts binding to active site does not get cuto Only one strand is used to load onto RISC—goal is to base pair 5’ end that is less stable is loaded Seed region= most strongly basepaired leads to cleavage Imperfect binding determines function leads to translation inhibition- Using the miRNA pathwayo Transfect siRNA that mimics the product of dicer processing to induce miRNA pathway- Recall Polio and 5’ capo miRNa avoids using 5’ cap to direct translation initiation and uses IRES (internal ribosome entry site) ribosome recognizes the complex stem loop structure and binds before AUG codon= IRESo cap dependent vs. cap independento repression by miRNA when 5’ cap and binding siteso cap with no sites no repressiono no cap with sites no repression miRNA is not inhibiting transcription, they are blocking early translation recruitment of cap- bypass eIF4E cap independent repressed cap recognitiono combinatorial control- Let-7 miRNA binds to 3’ UTR region of lin14o Several miRNAs can regulate a single mRNAo Lin41 has binding sites for
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