Metabolism Of Nuclear RNA - Nucleus.img.cas.cz

Metabolism of Nuclear RNA

Coding and non-coding RNAzzCoding RNAs (4% ) - transcriptomemRNAs : rapid turnoverhalf-life - few minutes (bacteria) few hours(eukaryotes)Non-coding RNAs (96% )rRNAs : 80% in actively dividing bacteriatRNAssnRNAs : included in mRNA processing (spliceosome),eukaryotes onlysnoRNAs : included in rRNA processing, eukaryotesonlymi-RNAs: RNA-induced transcription silencingRNA processingEnd-modifications : 7-methylguanosine cap, poly(A) tailSplicing : removal of intronsCutting events : rRNA, tRNA processingChemical modifications : addition of chemicalgroups, RNA editing

RNA Pol II is an RNA FactoryCapping of RNA pol IItranscriptszzzoccurs early in all RNA pol IItranscripts when the nascent RNAis in 20-35 nt stage (hnRNA, presnRNA)after cleaving the γ-phosphate(RNA-triphosphatase) a GMP isadded in inverted orientation (5 - 5 linkage) (RNAguanylyltranspherase)Terminal inverted guanosine ismethylated at the 7 position (7metyltranspherase)

Functions of CapzProtects mRNA from degradation by RNAseszEnhances splicing efficiency of mRNAszEnhances transport of mRNAs from nucleus to cytoplasmzIn the cytoplasm, the cap-structure is important for the initiation oftranslationThe eukaryotic translation initiation factor eIF4E binds directly to thecap-structuresnRNAs m7G - nuclear export, m3G - nuclear importzzPolyadenylationzzzzzzzRequires the sequence 5'-AAUAAA-3' plusthe sequence 5'-CA-3' 10-20 nt downstream, plusa 5'-(GU)-3' rich sequence further downstreamSix factors are needed (PAP, CPSF, CSF, CFI, CFII, PAB),Two stage reaction (cleavage and AMP addition)100-200 A residues added to give ‘poly(A) tail’mRNA encoding histones are not polyadenylated

Main steps in icity factor) binds AAUAAA inhnRNAz CStF (cleavage stimulatory factor)binds G/U rich sequence; CFI, CFIIbind in betweenz PAP (PolyA polymerase) binds &cleaves 10-35 b 3' to AAUAAA at slowratez PAP adds about 12 A residues slowly,CFI, CFII and CPSF fall offzzPABII (polyA-binding protein II)binds, now As are added rapidly untilreach 250, then stopsPurpose of PolyadenylationzzzzProtects mRNA – In cytoplasm poly(A) size decreases due toRNAses, however poly(A) polymerase continues rebuildingNecessary for translation (site of binding by poly(A)-binding protein I(PABI)Recruits mRNA to polysomesRequired for splicing out of the last intron

pre-mRNA splicingzz1.2.3.Introns are intervening sequences that “interrupt” eukaryotic genesand must be removed before uninterrupted exons coding forproteins leave the nucleus as mRNAThree types of intron are known:Group I introns - Found in organelle and bacterial genes along withsome lower eukaryotes (fungi, tetrahymena) nuclear genes Can self splice without the aid of proteins Require free GTP for splicingGroup II introns - Found in organelle and bacterial genes Can self splice without the aid of proteins Differ from Group I introns in sequence and mechanismNuclear introns - Found in eukaryotic nuclear genes Require proteins and other RNAs for splicingSelf splicing intronszzzzzS.Altmann and T. Cechgroup I requires binding of guanosine into active siteresults of splicing are mature RNA and linear intron RNAgroup II does not bind any nucleotide it uses activatedOH of adenosine in active in branch site.results of splicing are mature RNA and lariat intron

Splicing of nuclear intronszzzzzzRequires spliceosomeSpliceosome consists of snRNPs and at least 70 participatingproteinsTransesterifications are facilitated by base-pairing of snRNAsMost mRNA are derived from the corresponding pre-mRNATrypanosomas and Euglenoids construct mRNA from separateprecursors – trans splicingStandard intron boundary – ‘GU-AG’ rule

SR Proteins Direct Splicingby Defining ExonszzzzSR proteins family of 8 well conserved proteins.SR proteins have N-terminal RNP domains rich in serand arg.Function to align 5’ splice site and the branch pointby interactions with snRNP U1 and snRNP U2.Regulators of alternative splicing:

Alternative splicingWalter Gilbert - 1977z 40 - 60% of pre-mRNA transcriptsundergoes alternative splicingz Allow for tissue specific expressionz Immunoglobin genes, Drosophila sexdeterminationzTransport of mRNA: Balbiani rings

mRNA export and its alternate fates in the cytoplasmWhat is RNA Interference(RNAi)“The Process by which dsRNA silencesgene expression.” [Mittal, 2004]z Generally: Post transcriptional genesilencing (PTGS)z Results in : Degradation or translationinhibition: Heterochromatin formationz A field with many unknownsz

Handy RNAi TermsdsRNA: double stranded RNA, longerthan 30 ntz miRNA: microRNA, 21-25 nt.zz Encodedby endogenous genes.z Hairpin precursorsz Recognize multiple targets.zsiRNA: short-interfering RNA, 21-25 nt.z Mostlyexogenous origin.z dsRNA precursorsz May be target specificRNAi: Two Phase ProcesszInitiationz Generationzof mature siRNA or miRNAExecutionz Silencingof target genez Degradation or inhibition of translation

miRNA BiogenesiszzzTranscribed from endogenous gene as pri-miRNAz Primary miRNA: long with multiple hairpinsz Imperfect internal sequence complementarityCleaved by Drosha into pre-miRNAz Precursor miRNA: 70nt imperfect hairpinsz Exported from nucleusCleaved by Dicer into mature miRNAz 21-25ntz Symmetric 2nt 3’ overhangs, 5’ phosphate groupsNovina and Sharp, 2004siRNA BiogenesisDicer cleaves long dsRNA into siRNA 2125ntz dsRNA from exogenous sourcesz Symmetric 2nt 3’ overhangs, 5’phosphate groupsz Evidence for amplification in C. elegansand plantsz Allows persistence of RNAi?zNovina and Sharp, 2004

Protein machinery of RNAizDrosha - Processes pri-miRNA into pre-miRNA- Leaves 3’ overhangs on pre-miRNAzDicer- Cleaves dsRNA or pre-miRNA- Leaves 3’ overhangs and 5’ phosphategroupszRNA Dependent RNA Polymerase (RdRP) siRNA acts as primer for elongation ontarget mRNA – more dsRNA producedzArgonaute (Ago) - lies at the heart all RNAzRNA Induced Silencing Complex (RISC) - RNAizRNA induced transcriptional silencing (RITS) - RISC-silencing effector complexeseffector complexlike complex responsible for heterochromatin formation inS.pombe.

Proposed Biologic Roles‘Immune System’ of the Genome Antiviral Defense Suppress Transposon Activity Gene Regulation (Silencing)(e.g. MicroRNAs, Heterochromatin)RNAi ApplicationsGENETIC TOOLProbing Gene FunctionGENE THERAPYCombat Viral InfectionTreat Genetic Diseases(New expression strategies)

Setting up an RNAi experimentdesign of RNA oligosz siRNA synthesisz Introducing siRNA into the cellsz monitoring effectszDesign of specific siRNAzzzzMultiple siRNA to single targetConsider: Length: usually 19-23 nt (27-mers Kim et al. Nature Biotechnology, 2005),Content of G/C: (30-52%),Positions of A/U: in the sense strandPosition in target mRNA: at least 100bases downstream AUG,Avoid 5 or 3 UTR (Boese et al.,Methods in enzymology, 2005).Scrambled controlPositive control (GAPDH)

siRNA synthesiszChemical synthesiszIn vitro transcriptionzDigestion of long dsRNA by an RNase III family enzyme(e.g. Dicer, RNase III)zExpression in cells from an siRNA expression plasmid orviral vectorzExpression in cells from a PCR-derived siRNAexpression cassetteIntroducing siRNA into the cellsTransfectionz Electroporationz Concentration ranging from 5 to 50 nMz

Monitoring effectsqReal-Time PCRz Western blotz Immunofluorescencez Monitoring of interferon response: 2'5'oligoadenylate synthetase, dsRNAdependent protein kinase, PKRzThank you.

Coding and non-coding RNA zCoding RNAs (4% ) - transcriptome mRNAs : rapid turnover . RNA editing . RNA Pol II is an RNA Factory Capping of RNA pol II transcripts . Methods in enzymology, 2005). zScrambled control zPositive control (GAPDH) siRNA synthesis