hnRNA Processing I Capping Polyadenylation Lecture 5 Part A Features of Vertebrate mRNAs 5 end 5 cap 5 UTR Untranslated Region coding sequence exons speci ed in the gene assembly required 3 end put G residue on 5 end polyadenylated Poly A tail 3 UTR Untranslated NonCCoding Region polyadenyla on 5 cap 5 UTR exons poly A tail 3 UTR hnRNA Processing Capping PolyadenylaIon PREBmRNA PROCESSING Overview graphic of overview of preCmRNA processing hnRNA Processing Capping PolyadenylaIon RNA pol II transcripts are capped cap added within 30 nucleoIdes of CAPPING transcript iniIaIon CTD of RNA pol II binds capping RNA pol II transcripts get capped CTD of RNA pol II binds capping enzymes G gets added to transcript s 5 end 5 B5 triphosphate bond w purine 1 site cap contains methylated riboses bases enzymes heptad human CTD has 52 repeats of the YSPTSPS G nucleoIde added to 5 end of transcript G is in a 5 B5 triphosphate bond w purine 1 site cap has methylated riboses bases RNA pol II transcripts capped CTD binds capping enzymes G added to 5 end hnRNA Processing Capping Cap Synthesis 3 required acIviIes 3 PO4 s are already present in nascent RNA 5 CAP Synthesis 3 required acIviIes phosphatase guanyl transferase 2 methyl transferases 1 2 3 phosphatase removes Cphosphate 6 PO4 s only 3 get to stay 3 PO4 s come in w added G base guanyl transferase adds GMP postC transcripIonal 2 methyl transferases 1 forms N7Cmethylguanosine 2 methylates 2 COH of ribose in rst or second nucleoIde 3 acIviIes phosphatase guanyl transferase 2 methyl transferases hnRNA Processing Capping Cap Synthesis 5 CAP Major FuncUons mRNA stability protects mRNA from exoCnucleases deCcapping mRNA gets degraded splicing of preBmRNA 5 cap sImulates splicing hnRNP F binds 5 cap protein synthesis recognized by CBC sImulates protein synthesis 1 mRNA stability protects mRNA from exoCnucleases deCcapping associated w degradaIon of mRNA 2 splicing of preBmRNA 5 cap sImulates splicing hnRNP F Heterogeneous Nuclear Ribonucleoprotein F binds 5 cap depleIon of hnRNP F reduces splicing 3 protein synthesis recognized by CBC Cap Binding Complex CBC sImulates protein synthesis thus 5 cap also marks sequence for protein synthesis mRNA stability protecIon splicing of preCmRNA 5 cap sImulates protein synthesis CBC hnRNA Processing Capping FuncIons vertebrate mRNAs have poly A POLYB ADENYLATION 3 end poly A found 10C30 bps downstream of AAUAAA 200C300 A s not encoded in DNA just added on to DNA RNA pol I RNA pol III transcripts mRNAs have poly A on 3 end poly A downstream of AAUAAA A s not encoded in not polyadenylated DNA RNA pols I III transcripts RNA pol II some mRNAs lack poly A histone mRNAs of RNA pol II not polyadenylated but most have it histone mRNAs not polyadenylated poly A 3 end poly A A s not encoded in DNA hnRNA Processing PolyadenylaIon consensus sequence for poly A POLYB ADENYLATION Consensus addiIon 1 AAUAAA 2 downstream sequence rich in U s or GBU s CA gets cleaved UC or GBUCrich gets recognized cleavage site frequently CA consensus sequence for poly A addiIon AAUAAA downstream rich in U s or GBU s consensus AAUAAA downstream rich in U s or UCG s hnRNA Processing PolyadenylaIon Consensus POLYB ADENYLATION Factors sUmulaUon factor ST factor speci city factor SP factor endoribonuclease CF poly A polymerase PAP sUmulaUon factor speci city factor endoribonuclease poly A pol requires ATP for acIon does nonCtemplate addiIon of A s in polyadenylaIon requires ATP for poly A pol acIon sImulaIon factor speci city factor endoribonuclease poly A pol needs ATP hnRNA Processing PolyadenylaIon Factors POLYADENYLATION Factor Recruitment CStF Cleavage StimulaIon Factor CPSF Cleavage PolyadenylaIon Speci city Factor RNA Pol II TFIID Bind PolyadenylaUon Factors TFIID binds CPSF CTD of RNA pol II binds CStF CPSF factors eventually transferred to preCmRNA CStF CPSF hnRNA Processing PolyadenylaIon Factor Recruitment Steps in Poly A Synthesis CPSF recognizes binds this sequence AAUAAA POLYADENYLATION Factor Assembly Assembly of Factors for Poly A Synthesis 1 CPSP binds AAUAAA 2 CStF associates w GCUCrich region CStF 3 Cleavage factors bind CPSF 4 Poly A pol PAP is recruited into protein mRNA complex 5 RNA is nicked by cleavage factors CPSP CStF cleavage factors bind CPSF CStF PAP RNA nicked hnRNA Processing PolyadenylaIon Cleavage Extension conCnues on next slide POLYADENYLATION Factor Assembly conCnued from last slide AddiUon of Poly A to PreBmRNA 1 PAP adds A s to 3 COH oligoBA up to 10 nucleoIdes added CPSP acIvates PAP PABPII Poly A Binding Protein II binds oligoBA tail PAP rapidly extends oligoBA tail into poly A tail 2 3 PABPII falls o when it enters cytoplasm mRNA in cytoplasm has PABPI PAP adds oligoCA tail 3 COH PABPII binds oligoCA PAP extends oligoCA tail hnRNA Processing PolyadenylaIon mRNA Stability POLY A TAIL FuncUons 1 reduces degradaIon of mRNA PABPI Poly A Binding Protein I binds poly A tail PABPI might protect mRNA from nucleases i e increase halfClife of mRNA loss of poly A tail associated w mRNA degradaIon reduces degradaIon of mRNA PABPI binds poly A tail PABPI protects mRNA from nucleases poly A tail loss associated w mRNA degradaIon PABPI binds poly A tail PABPI protects mRNA from nucleases hnRNA Processing PolyadenylaIon Poly A Tail FuncIons POLY A TAIL FuncUons sImulates protein synthesis postCtranscripIonal control of gene expression sImulates protein synthesis 2 3 postCtranscripIonal control of gene expression situaIon mRNA in cell but no protein or very liile produced signal received during di erenIaIon results in cytoplasmic poly A addiIon facilitates translaIon of mRNA PABPI binds poly A tail in cytoplasm w o IFs iniCaCon factors mRNA might get degraded development ajer ferIlizaIon eggs oocytes sImulates protein synthesis postCtranscripIonal control of gene expression hnRNA Processing PolyadenylaIon Poly A Tail FuncIons POLYADENYLATION Quality Control on mRNA Processing improperly processed mRNA degraded by the exosome nuclear export cannot proceed if mRNA improperly processed might get degraded but also can t leave the nucleus nonCsenseCmediated decay mRNA w premature STOP degraded mRNA degradaIon can proceed from 5 cap poly A tail or middle mRNA is very vulnerable so cell has lots of mechanisms to protect it circumstances when mRNA degradaIon occurs hnRNA Processing PolyadenylaIon Quality Control on mRNA Processing