Understanding RNA: Structures, Functions, and Translation Control

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University of British Columbia**We aren't endorsed by this school

Course

BIOC 410

Subject

Biology

Date

Dec 12, 2024

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Uploaded by PrivateWater37729

Nov 25 2024Biochemistry 410 - RNA SectionLearning objectives:Coding and Non-Coding RNAs, How to detect RNAs?Secondary and tertiary structures of RNA and how to study themRNA-based enzymes: ribozymes (e.g., the ribosome) – the ‘RNA world’RNA molecules as sensors: riboswitchesRNA processingThe control of mRNA translationThe regulation of the translation of specific mRNAsFunctional screens (RNA interference/CRISPR)(Epitranscriptome)Dr. Eric JanProfessorLife Sciences Building, 5.457ej@mail.ubc.caBIOC410 Office hoursNov 25 3:30-4:15 pmDec 2 3:30-4:15 pmDec 5 10:00-10:45 am

Nov 25 2024Learning Objectives Lecture 7- Decoding codon:anticodon- aminoglycosides – how they lead to miscoding-aminoacylation – accuracy of the correct amino acid and tRNA selectionmicroRNA/siRNA

Nov 25 2024Stereo views of the codon-anticodon base pairsBase pair 1Base pair 2Base Pair 3Base Pair I and II forms hydrogen bonds that sensesthe presence of both bases (which are in a WC base pair)Base Pair III only interacts withone of the basesThis anticodon-codondiscrimination process leads to the next step in translation

Nov 25 2024Over 50% of antibiotics known target the ribosome!Some specifically target bacteria, some targetboth bacteria and eukaryotic ribosomes

Nov 25 2024Mechanism of action of neomycin and hygromycin Bneomycin- causes miscoding, increases cognate or non-cognate A-site tRNA bindinghygromycin B- inhibits translocation, tRNA movementFlip outand recognizecognatecodon:anticodonminor grooveClass of antibiotics:aminoglycosides

Nov 25 2024Cognate anticodon-codon decoding leads to conformational changes in the 30S, tRNA and EF-TU Resulting in GTP hydrolysisAfter GTP hydrolysis, EF-Tu-GDP dissociates andthe aminoacyl-tRNA undergoes accommodation(from purple to blue)

Nov 25 2024tRNAs are aminoacylatedHow does the correct amino acid find the correct tRNA?All tRNAs adopt the same structure, is it just the anticodon?AcceptorstemAnticodonloop

Nov 25 2024Aminoacylation of tRNAs- distinct 20 aminoacyl-tRNA synthetases catalyze the addition of an aminoacyl group to the CCA end of a tRNA (MBOC 5th Ed. pp. 1082)- Class I (e.g. IleRS, ValRS, LeuRS) and Class II (e.g.ThrRS, AlaRS, ProRS)Two steps are involved1) ATP + amino acid aminoacyl-AMP + PPi2) aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

Nov 25 2024How does an Alanine tRNA synthetase recognize the Ala-tRNA?A3C70Conserved Nucleotidesgray shading- alterations do not affect aminoacylationMeasure [3H]-Alaaminoacylation usingpure Ala-tRNA synthetase

Nov 25 2024U3G70Conserved Nucleotidesgray shading- alterations do not affect aminoacylationHow does an Alanine tRNA synthetase recognize the Ala-tRNA?Measure [3H]-Alaaminoacylation usingpure Ala-tRNA synthetase

Nov 25 2024Conserved Nucleotidesgray shading- alterations do not affect aminoacylationC6G7G67C66G3 and U70 are necessaryfor Alanine aminoacylationHow does an Alanine tRNA synthetase recognize the Ala-tRNA?Measure [3H]-Alaaminoacylation usingpure Ala-tRNA synthetase

Nov 25 2024Conserved Nucleotidesgray shading- alterations do not affect aminoacylationblue- nucleotidesthat are differentfrom Ala-tRNAMeasure [3H]-Alaaminoacylation usingpure Ala-tRNA synthetaseG3 and U70 are necessary and sufficient for Alanine aminoacylationHow does a tRNA synthetase recognize the right tRNA?

Nov 25 2024How do specific aminoacyl-tRNA synthetases recognize the correct tRNA?- aminoacyl-tRNA synthetases recognize specific residues within the acceptor stemand the anticodon stem called Identity Elements- recognition is specific to the tRNA and sometimes does not require the anticodon(in the Ala tRNA, all you need is the acceptor stem!)anticodonanticodonstemacceptor stemboundATP(active site)

Nov 25 2024Identity Elements

Nov 25 2024Aminoacyl-tRNA synthetases contain multiple proofreading steps- charging of tRNAs is highly accurate: error rate is 1 in 40,000How? Example: Isoleucyl-tRNA synthetase (IleRS)- can discriminate isoleucine 3000 fold over valineOnly a methylene difference!- a subset of aminoacyl-tRNA synthetasescontains an editing domain, which canhydrolyze misacylated aminoacyl-tRNAs- editing domain is distinct of the activesynthetase domain- some editing domains are in trans!(e.g. TyrRS, AlaRS)Double Sieve Model:TyrValIleValValIleIleIleIleStep 1Post-Step 2:editing domainRecent data also, show that the Val-AMPsubstrate is hydrolyzed too!

Nov 25 2024tRNAeditingdomainanalogof Ilein active sitecorrectacylated tRNAforms a hairpinmisacylatedtRNAAminoacyl-tRNA synthetases contain multiple proofreading stepsScience 285, 1074 (1999);DOI: 10.1126/science.285.5430.1074

Nov 25 2024Questions: You are interested in the RNA world hypothesis. It has been hypothesized thatThe ribosome may be a molecular fossil of the RNA world hypothesis. What statements are true or false?1)The catalytic activity of the ribosome is carried out by ribosomal RNA (rRNA), not proteins.2) Peptidyl-transferase of the ribosome involves the 2’OH of the ribose of the incoming ribosomal A site aminoacyl-tRNA to align with the the amine of the 2’OH of the ribose of the ribosomal P site peptidyl-tRNA. 3) The ribosome's structure is highly conserved across all domains of life, suggesting ancient origins.4) The RNA world hypothesis proposes that RNA once served as both genetic material and a catalyst.5) Prokaryotic tRNAs can be used for translation with eukaryotic ribosomes.

Nov 25 2024Biochemistry 410 - RNA SectionLearning objectives:Coding and Non-Coding RNAs, How to detect RNAs?Secondary and tertiary structures of RNA and how to study themRNA-based enzymes: ribozymes (e.g., the ribosome) – the ‘RNA world’RNA molecules as sensors: riboswitchesRNA processingThe control of mRNA translationThe regulation of the translation of specific mRNAsFunctional screens (RNA interference) - microRNAs(Epitranscriptome)Dr. Eric JanProfessorLife Sciences Building, 5.457ej@mail.ubc.caBIOC410 Office hoursNov 25 3:30-4:15 pmDec 2 3:30-4:15 pmDec 5 10:00-10:45 am

Nov 25 2024RNA-mediated gene silencing: Highlights1990/1992transcription of transgene can reduce expression of RNAstermed Post-transcriptional Gene Silencing (PTGS)1993small 21 nt RNA called lin-4 represses translation of lin-14in C. elegans1995injection of sense and antisense transcripts produced thesame knockdown of specific gene expression (Kemphues)1998Andy Fire and Craig Mello discovered that dsRNA-mediatedsilencing in C. elegans, very little dsRNA required1999Zamore/Tuschl groups found that dsRNA were 'diced' into 21 nt RNAs2001-Discovery of hundreds of miRNAs in humans, flies, C. elegansetc...

Nov 25 2024Discovery of many microRNAs- 21-23 nucleotide endogenous noncodingRNAsScience, 2001- >24,000 miRNAs ranging from virus, plant, insect to animals (microrna.sanger.ac.uk/sequences) updated >38,589!- 1917 entries for human miRNAs- about 1/3 conserved across species and 60% of miRNA loci are conserved mouse/man

Nov 25 2024miRNA Biogenesispri-miRNAProcessed byDrosha~35% of miRNAscome from introns!mirtrons~70 nt pre-miRNAExported by Exportin 5mature 21-22 ntdsRNA with 2 nt3’ overhangsone of the strandsis selected and bound by RISCEndonucleolytic CleavageTranslational RepressionDicerRISC has at leastone Ago, only Ago2 has cleavage activitymost miRNAsSmall interferingRNAs- foreign dsRNA- viral dsRNA- experimental

Nov 25 2024Loss of Luciferase activity Asymmetric strand selection with miRNA and siRNA5'-cacguacgcggaauacuucga-3'5'-uuucgaaguauuccgcguacg-3'orreporter geneluciferaseluciferin substrateMeasure luciferase activityby light emissionLoss of Luciferase activity isreflective of cleavage activitywill hybridize towill hybridize toTherefore, black siRNA is the guide (mature) strand and the red siRNA is the passenger strand. Incubate in Drosophila embryo extractsdsRNAs that can hybridize

Nov 25 2024Asymmetric strand selection with miRNA and siRNAreporter gene5'-cacguacgcggaauacuucga-3'5'-uuucgaaguauuccgcguacg-3'orluciferaseluciferin substrateMeasure luciferase activityby light emissionIncubate in Drosophila embryo extractsdsRNAs that can hybridizeLoss of Luciferase activity is reflective of cleavage activityLoss of Luciferase activity Luciferase activityGU bp wobble, lower free energy than AU bp atthe other endNow the red siRNA isthe guide strand andthe black is the passenger strandThe more unstable 5' end of the duplex, the 5' strand becomes the guide strandLoss of Luciferase activity

Nov 25 2024Filipowicz et al., 2008 miRNA maturation- miRNAs can be processed from pol II transcripts within introns and as clustersof miRNA hairpins (and likely within exons too)- asymmetric strand selection for RISC loading is based on the stability of the5'end of the duplex mRNA targeting by miRNAKey concept: seed region has to be fully complementary for miRNA and siRNA activity- 3' to the seed region may contain bulges or loops that are likely importantfor activity

Nov 25 2024Filipowicz et al., 2008 mRNA targeting by miRNAHow is miRNA binding to mRNA repressing translation?Level of initiation or elongation?Key concept: seed region has to be fully complementary for miRNA and siRNA activity- 3' to the seed region may contain bulges or loops that are likely importantfor activity

Nov 25 2024OD 254 nM40S60S1 2 3 4 5 6 7 8 9 10 115’3’10%50%Sucrose gradient40S60SLow molecular- weight fractionsHigh molecular- weight fractionsPolysome Analysis using Sucrose Gradient Density Centrifugation80SpolysomesTopBottomrRNA represents 95% oftotal RNA in the cell

Nov 25 2024How do miRNAs repress translation?Six imperfect binding sites to a miRNAcalled CXCR4 (artificial)TransfectedsiRNA/miRNAbinding siteswithin 3'UTRTransfection of CXCR4 miRNA leads totranslational repressionpolysomesNo change inpolysomaldistribution ofreporter RNAmiRNA repressestranslation atthe elongationstep

Nov 25 2024Reporter RNA is repressedat the translational level due tolet-7 binding sites in 3'UTR-Transfect plasmids that expressreporter RNA in Hela cells- Hela cells express endogenous let-7 miRNAHow do miRNAs repress translation?let-7 miRNA polysomesShift to lighterfractions, lessribosomes associatedTherefore, miRNA-dependent translationalrepression is at the initiation level

Nov 25 2024Models for miRNA-mediated translational repressionAgo-2 implicated in binding to capInhibition of 60S joiningInhibition of elongationProteolysis of nascent protein Deadenylation

Nov 25 2024Each miRNA can regulate hundreds of target mRNAs3'UTRs may containmultiple miRNAbinding sites- algorithms predict that miRNAs regulate between 30-95% of gene expression

Nov 25 2024How to detect miRNAs or miRNA activity?To measure miRNA levels- Northern blot analysis- RT-PCR-microarray analysis-NGS-seqRNA from differentcancer cell linesNormalcellsHow to detect miRNA activity?- use reporters that has a binding sitein its 3'UTR- can be perfect or imperfectcomplementaritypri-

Nov 25 2024miRNA Biogenesispri-miRNAProcessed byDrosha~35% of miRNAscome from mirtrons!mirtrons~70 nt pre-miRNAExported by Exportin 5mature 21-22 ntdsRNA with 2 nt3’ overhangsone of the strandsis selected and bound by RISCEndonucleolytic CleavageTranslational RepressionDicerRISC has at leastone Ago, only Ago2 has cleavage activitymost miRNAsSmall interferingRNAs- foreign dsRNA- viral dsRNA- experimental

Nov 25 2024Using RNAi to “knock down” expressionshRNA = shorthairpin RNALoss of specific mRNA: eventually, loss of specific protein (test by Western blot) might have to wait for daysHairpinTandemTheoretically, you can knock down expression ofany mRNA (or RNA) specificallycleavage is catalyticDirect transfectionof siRNAs(e.g. Dharmacon)Can use RNA PolIor PolII -drivenpromoters

Nov 25 2024RNAilibrariesIdentifies a ‘new’ geneas being involved in thephenotype under studyCells seeded in plateDifferent RNAi fromlibrary in each wellScreen for phenotypeof interest

Nov 25 2024Genome-wide siRNA screens to identifyhost genes involved in viral replication- knock down expression of individual genes(target 21,121 human genes or if in Drosophilacells, target 13,071 genes ~90%)- add virus (HIV, West Nile Virus, Influenza,Drosophila C virus) - identify those genes that facilitate or suppress viral replicationAlthough HIV only encodes for 15 proteins,identified many HIV-dependency hostgenes Identify antiviralgenesIdentify host genesimportant for virusinfectionAntiviral therapy

Nov 25 2024Target Viruses directly with dsRNA- target poliovirus with siRNAs- interestingly, could find siRNA-resistant polioviruses- found mutations in the poliovirusgenome within the target sequence of the siRNA(seed sequence was mutated)- target the HIV genome (gag protein), the cellular receptor for HIV, CD4 New therapeutic strategy againstviruses, cancer, and other diseasesXsiRNA to poliovirusresistant poliovirus