Molecular biology - Chapter 15: Messenger rna processing II: Capping and polyadenylation

Molecular BiologyFourth EditionChapter 15Messenger RNA Processing II: Capping and PolyadenylationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.115.1 CappingBy 1974, mRNA from a variety of eukaryotic species and viruses were found to be methylatedA significant amount of this methylation was clustered at the 5’-end of mRNAThis methylation cluster formed a structure we call a cap2Cap StructureEarly study used viral mRNA as they are easier to purify and investigateThe b-phosphate of a nucleoside triphosphate remains only in the first nucleotide in an RNACap is at the 5’-terminus of RNAThe cap is made of 7-methylguanosine, m7GLinkage is a triphosphateCharge on the cap area is near -53Reovirus Cap StructureThe m7G contributes a positive chargeTriphosphate linkage contributes 3 negative chargesPhosphodiester bond contributes 1 negative chargeTerminal phosphate contributes 2 negative charges4Cap SynthesisFirst step RNA triphosphatase removes terminal phosphate from pre-mRNAThen, guanylyl transferase adds capping GMP from GTPNext, 2 methyl transferases methylate N7 of capping guanosine and 2’-O-methyl group of penultimate nucleotideThis occurs early in transcription, before chain is 30 nt long5Functions of CapsCaps serve at least four functions:Protect mRNAs from degradationEnhance translatability of mRNAsTransport of mRNAs out of nucleus Efficiency of splicing mRNAs615.2 PolyadenylationThe process of adding poly(A) to RNA is called polyadenylationA long chain of AMP residues is called poly (A)Heterogeneous nuclear mRNA is a precursor to mRNA7Poly(A)Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nt long at their 3’-endsPoly(A) is added posttranscriptionally by poly(A) polymerase8Functions of Poly(A)Poly(A) enhances both the lifetime and translatability of mRNARelative importance of these two effects seems to vary from one system to anotherIn rabbit reticulocyte extracts, poly(A) seems to enhance translatability by helping to recruit mRNA to polysomes9Basic Mechanism of PolyadenylationTranscription of eukaryotic genes extends beyond the polyadenylation siteThe transcript is: CleavedPolyadenylated at 3’-end created by cleavage 10Synthesis and Polyadenylation11Polyadenylation SignalsAn efficient mammalian polyadenylation signal consists of:AAUAAA motif about 20 nt upstream of a polyadenylation site in a pre-mRNAFollowed 23 or 24 bp later by GU-rich motifFollowed immediately by a U-rich motifVariations on this theme occur in natureResults in variation in efficiency of polyadenylationPlant polyadenylation signals usually contain AAUAAA motifMore variation exists in plant than in animal motifYeast polyadenylation signals are even more different12Cleavage of Pre-mRNAPolyadenylation involves both:Pre-mRNA cleavagePolyadenylation at the cleavage siteCleavage in mammals requires several proteinsCPSF – cleavage and polyadenylation specificity factorCstF – cleavage stimulation factorCF ICF IIPoly (A) polymeraseRNA polymerase II13Initiation of PolyadenylationShort RNAs mimic a newly created mRNA 3’-end can be polyadenylatedOptimal signal for initiation of such polyadenylation of a cleaved substrate is AAUAAA followed by at least 8 ntWhen poly(A) reaches about 10 nt in length, further polyadenylation becomes independent of AAUAAA signal and depends on the poly(A) itself2 proteins participate in the initiation processPoly(A)polymeraseCPSF binds to the AAUAAA motif14Elongation of Poly(A)Elongation of poly(A) in mammals requires a specificity factor called poly(A)-binding protein II (PAB II)This protein Binds to a preinitiated oligo(A)Aids poly(A) polymerase in elongating poly(A) to 250 nt or morePAB II acts independently of AAUAAA motifDepends only on poly(A)Activity enhanced by CPSF15Polyadenylation ModelFactors assemble on the pre-mRNA guided by motifsCleavage occursPolymerase initiates poly(A) synthesisPAB II allows rapid extension of the oligo(A) to full-length16Poly(A) PolymeraseCloning and sequencing cDNAs encoding calf thymus poly(A) polymerase reveal a mixture of 5 cDNAs derived from alternative splicing and alternative polyadenylationStructures of the enzymes predicted from the longest sequence includes: RNA-binding domainPolymerase module2 nuclear localization signalsSer/Thr-rich region – this is dispensable for activity in vitro17Turnover of Poly(A)Poly(A) turns over in the cytoplasmRNases tear it downPoly(A) polymerase builds it back upWhen poly(A) is gone mRNA is slated for destruction18Cytoplasmic PolyadenylationCytoplasmic polyadenylation is most easily studied using Xenopus oocyte maturationMaturation-specific polyadenylation of Xenopus maternal mRNAs in the cytoplasm depends on 2 sequence motifs:AAUAAA motif near the end of mRNA Upstream motif called the cytoplasmic polyadenylation element (CPE)UUUUUAUOr closely related sequence1915.3 Coordination of mRNA Processing EventsAfter reviewing capping, polyadenylation and splicing, it is clear that these processes are relatedCap can be essential for splicing, but only for splicing the first intronPoly(A) can also be essential, but only for splicing out the last intron20Effect of Cap on SplicingRemoval of the first intron fro model pre-mRNAs in vitro is dependent on the capThis effect may be mediated by a cap-binding complex involved in spliceosome formation21Effect of Poly(A) on SplicingPolyadenylation of model substrates in vitro is required for active removal of the intron closest to the poly(A)Splicing any other introns out of these substrates occurs at a normal rate even without polyadenylation 22mRNA-Processing Occurs During TranscriptionAll three of the mRNA-processing events take place during transcriptionSplicing begins when transcription is still underwayCapping When nascent mRNA is about 30 nt longWhen 5’-end of RNA first emerges from polymerasePolyadenylation occurs when the still-growing mRNA is cut at the polyadenylation site23Binding of CTD of Rpb1 to mRNA-Processing ProteinsThe CTD of Rpb1 subunit of RNA polymerase II is involved in all three types of processingCapping, polyadenylating, and splicing enzymes bind directly to the CTD which serves as a platform for all three activities24CTD PhosphorylationPhosphorylation state of the CTD of Rpb1 in transcription complexes in yeast changes as transcription progressesTranscription complexes close to the promoter contain phosphorylated Ser-5Complexes farther from the promoter contain phosphorylated Ser-2Spectrum of proteins associated with the CTD also changesCapping guanylyl transferase is present early when the complex is close to promoter, not laterPolyadenylation factor Hrp1 is present in transcription complexes near and remote from promoter25RNA Processing Organized by CTD26Coupling Transcription Termination with End ProcessingAn intact polyadenylation site and active factors that cleave at the polyadenylation site are required for transcription terminationActive factors that polyadenylate a cleaved pre-mRNA27Mechanism of TerminationTermination of transcription by RNA polymerase II occurs in 2 steps:Transcript experiences a cotranscriptional cleavage (CoTC) within termination region downstream of the polyadenylation siteThis occurs before cleavage and polyadenylation at the poly(A) siteIt is independent of that processCleavage and polyadenylation occur at the poly(A) siteSignals polymerase to dissociate from template28Termination SignalCoTC element downstream of the polyadenylation site in the human b-globin mRNA is a ribozyme that cleaves itselfThis generates a free RNA 5’-endThis cleavage is required for normal transcription terminationIt provides an entry site for Xrn2, a 5’3’ exonuclease that loads onto the RNA and chases RNA polymerase by degrading the RNA29Xrn2, ExonucleaseXrn2 terminates transcription like a “torpedo”There is a similar torpedo mechanism in yeast where cleavage at poly(A) site provides entry for the 5’3’ exonuclease Rat1Rat1 degrades the RNA until it catches the polymerase and terminates transcription30Torpedo Model for Transcription Termination31Role of Polyadenylation in mRNA TransportPolyadenylation is required for efficient transport of mRNAs from their point of origin in the nucleus to the cytoplasm32