BIO 344 1st Edition Lecture 18 Outline of Last Lecture I. Translational controla. Amino acid starvationII. Global translation down regulated in amino acid starvationa. yeasti. Gcn2 kinase and eIF2 on Gcn4ii. Ternary complex on site selectionb. Mammalsi. HRI, PKR, PERK, Gcn2ii. Mice food selectioniii. Alcohol derivativesOutline of Current 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 Current LectureRNAi- RNAi= interference RNAo plants use as an antiviral mechanismThese 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.o horticulture industry attempted to use to make new/vibrant flower colors but instead accidentally lead to discover of RNAi and function RNAse protection assay Endogenous is suppressed by transgene- Post transcriptionally- C. Eleganso Par1= kinase important in polarity in embryoso Antisense RNA= interferes with translation by base pairing with mRNA and physically clocking translational machinery Stoichiometric must be sitting at the site to block, NOT catalytico Injected antisense and sense RNA to see what happens to development Minus RNA +Par1 protein Antisense RNA - Par1 protein Sense RNA -Par1 protein- You’d expect no effect here because it can not base pair with the start codon- Still get an effect like antisense because of contamination of dsRNA—more on dsRNA effect in Unc54 experiment explanationo Unc54= uncoordinated—myosin-like, essential for movement When mutated-- twitching/paralyzed, no movement Injected complementary RNA to various introns and econs into adult gonad and assayed in F1 progeny- F= promoter, D and E= intron, A and B= exon of unc54- For unc54A:o Sense RNA on exon 6 100% WTo Antisense on exon 6 100% WTo Sense and antisense on exon 6100% paralyzed Sense and antisense basepair with eachother—dsRNA- For Unc54B: same results- For Unc54 F, D, and Eall 100% WT- This tells us that because exons are only affected, must occur post transcriptionally—in cytoplasm- Also found that it is possible for these effects to persist in 2nd and maybe 3rd generationo RNAi is different than antisense—it is catalytic rather than stoichiometric RNAi is most efficient, greatest response is elicited, when dsRNA (sense-antisense) is presentRNAi Mechanism- dsRNA of significant length (~300-550 bp) is taken up by cells and cleaved by dicer enzymeo dicer recognizes long dsRNA and chops at discrete length of 20-30 nucleotides long fragments (2 helix turns), resulting in short dsRNAo 2 nucleotide 3’ overhangs produced  Dicer has 2 RNAse III-like domains that cut staggered ends- short dsRNA bind to RISC (RNA-induced silencing complex)o RISC complex= Argonaut protein and silencing RNA (siRNA/miRNA)o Argonaut= catalytic cleaving protein Recognizes RNAs by length and staggered ends RISC binds and cleaves short dsRNA- One cleaved RNA strand falls out of active site- siRNA=>Other strand now remains bound to RISC and serves as a search probe to link RISC to mRNA- RISC + siRNA (guide) = cleavage/degradation of mRNA- So, Catalytic target mRNA cleavagedegradationgene silencingAntiviral Pathway in plants- During viral infection, RISC gets programmed with siRNAs for viruses that will silence viral gene- Used GFP to assay for RNAi effects in subsequent generationso GFP served as “virus” and gradually was suppressed completely after several generationso GFP gene was made in tandem with a promoter—making a sense and antisense complementary RNA folded into a long double stranded hairpin structureo Chopped by dicer, RISC recognizes and cleaveso Systemic spread that allows this response to continue until no GFP caused by amplified signal RNA dependent RNA polymerases amplify the signal- See slide imagesHuman Pathway- In humans, long dsRNA does NOT naturally induce RNAi response - dsRNA signals apoptosis= programmed cell deatho induced by phosphorylation of EIF2 alpha by a kinase related to Gcn2 recall that Gcn2 recognized aa tRNA synthetase-like  this kinase recognizes dsRNA instead- long dsRNA activates PKR to shut down translation- how can we avoid triggering PKR?o Short pre cleaved RNA can be inserted Looks like it has been processed by dicer (we have dicer and RISC naturally) Now siRNA will do work rather than PKR being activatedUsing RNAi in forward genetic screen for host factors- The host proteins previously reported to facilitate West Nile virus (=host susceptibility factors, HSFs) comprise endosomal transport regulators and vATPase (for entry), eEF1A, TIA-1/TIAR and HMGCR (for replication), and c-Yes (for secretion)- Other host proteins may reduce WNV infection (termed host resistance factors, HRFs): components of the antiviral IRF3 pathway are known HRFs of WNV infection11- performed a genome-scale siRNA-based screen silencing 21,121 human genes in HeLa cells to comprehensively identify the cellular proteins associated with the early stages of WNV infection, from viral entry through to the intracellular translation of viral RNA- assay involved infection of gene-silenced cells with WNV for 24 h, followed by a microscopy-based quantification of the cells immunostained for viral envelope protein to select the candidate host proteins- The screen was done in two stepso a primary screen using a pool of four siRNAs per geneo a validation screen, testing each individual siRNA within the pool separately (for the hits selected in the primary screen) to minimize potential off-target