1Lecture #16 Processing of viral pre-mRNA(Chapter 10)In order for viral protein to be synthesized viral mRNAs aretranslated by cellular protein synthetic apparatus (Lecture #15)Viral mRNAs haveevolved to be very stable.Viral mRNAs must conform to the requirements of host cell translation system1) RNA processing- a series of covalent modifications.2) Viral mRNAs produced in the nucleus must be exported to the cytoplasm.3) Once in the cytoplasm, gene expression is a balance between the translatability of anmRNA and its stability.123Fig 10.1Pre-mRNA modification of cellular mRNAs is performed inthe nucleus– Addition of 5’ 7Methyl-Gppp caps– Addition of 3’ poly-adenylated tails– Splicing– RNA editing (in some cases)-introduction of nucleotides thatare not specified by a cellular or viral gene.COVALENT MODIFICATIONS DURINGVIRAL PRE-mRNA PROCESSING2Capping of cellular pre-mRNA 5’ ends•5’ 7MeGppp caps discovered using reovirus &vaccinia virus.Cellular mRNAs capped cotranscriptionally inthe nucleus by action of 5 enzymesFunctions of the cap:–Protect 5’ end from exonucleolytic attack–Interact with translation initiation apparatus–Mark mRNAs as “selfFig. 10.2Capping enzyme1. Synthesized by host cell enzymes - Retroviruses, Adenoviruses2. Synthesized by viral enzymes - Poxviruses, Reoviruses.3. Cap snatching: virus steal caps from host mRNAs. - Influenza.• Some RNA viruses have evolved around requirement for cap.– Proteins covalently bound to 5’ end substitute for caps. e.g. Picornaviruses– Translation initiated internally on mRNA at IRES elements. e.g. HepatitisC virus.Capping of viral pre-mRNA 5’ endsTable 10.1Synthesis of 3’ polyA tails: cellular mRNAs.• PolyA tails also first discovered using viralsystems.• All cellular mRNAs have non-templatedpolyA tails attached to their 3’ ends.• PolyA tails are post-transcriptionally added tocellular mRNAs using a series of cis-actingsequences on the mRNA and trans-actingribonucleoprotein factors• PolyA tails interact with PolyA-bindingprotein: important for translation.(Fig. 10.3)3Synthesis of 3’ polyA tails: viral mRNAsViral mRNAs can be polyadenylated by host or viral enzymes.1. Host enzymes: Occurs like host mRNAs. –Post-transcriptionally–Examples: retroviruses, herpesviruses, adenoviruses.Synthesis of 3’ polyA tails: viral mRNAsFig 10.4Alternative:dispensed with polyA tails altogether.Use complex 3’ mRNA structures to interact withpolyA-binding protein2. By Viral enzymes•Can occur post-transcriptionally.•Example: poxviruses•Can occur co-transcriptionally:–Copying of a long polyU stretch in template RNA:picornaviruses, M virus of yeast–Reiterative copying of short U stretches in templateRNA: Ortho- and ParamyxovirusesDiscovery of splicing of pre-mRNA Observation: heterogeneous nuclear RNA(hnRNA)–Larger than mRNA–Has same 5’ and 3’ UTRs as mRNAConclusion: both sides are preserved in mRNA but somehowinformation in-between is lost4Splicing of pre-mRNASharp and Roberts (1993 Nobel Prize).•The Adenovirus late major mRNA.•Contains sequence derived from 4 different blocks of genomicsequence.•Precursor nuclear major late RNA has the 4 sequence blocks +all sequence in between.Conclusion: in between sequences are “spliced out” of the pre-mRNA to make the mature mRNA.Mechanisms of splicingSplice sites: short consensussequences at 5’ and 3’of intron.5’: GU3’: AGFig 10.5Splicing must be completed with high degree ofaccuracy to ensure coding information is not lost.Regulated by cellular proteins and RNAs.Spliceosome: The large complex that assembles on anintron-containing pre-mRNA prior to splicing.snRNA; small nuclear RNAs (U1, U2, U4, U5 & U6)snRNP: small nuclear ribonucleoproteins. snRNAsassociated with proteins.Splicing: evolutionary implications•Exons contain protein coding information.•Shuffling of exons can be used to create new functionalarrangements.•Reflected in modular arrangement of many proteins.•Introns facilitate transfer of genetic information betweencellular and viral genomes5• Constitutive splicing:– Every intron is spliced out; Every exon is spliced in.• Alternative splicing:– All introns spliced out; Only selected exons spliced in.– Result: mRNAs having different coding information derived from a single gene.•(Fig. 10.8)Constitutive vs. Alternative splicingFig 10.8Alternative splicing and viruses•Allows expansion of the limited coding capacity of viral genomes•Can be employed to temporally regulate viral gene expression•Can control balance in the production of different regulatory units.•Can control balance in production between spliced and unspliced RNAs.Fig. 10.9SV40- early pre-mRNA•Splice at 1 of 2 suboptimal 5’ splicesites.•mRNAs encoding large T or small Tantigen.•Regulated by Sf2-splicing factor 2 .Alternative splicing and virusesFig. 10.11Retroviral gene expression:Unspliced RNAs:–Encode 5’ genes (gag-pol)–Can be used as ‘genomes’ for packaging inside of nascent viral particlesSpliced RNAs:-mRNAs encoding 3’ information (env gene)Balance in splicing is critical to optimal virus production6Fig 10.13RNA editingCotranscriptional editing•Occurs only in mRNA encoding the P protein (Paramyxoviridae-measles )•Polypyrimidine run at editing site.•Results in slipping backward one (-1) or two (-2) positions.•Changes translational reading frame.Posttranscriptional editing• Utilizes RNA adenosine deaminase.•Deaminates adenine bases to inosine•Converts UAG (stop codon) to UGGRNA editingFig. 10.14Nuclear ExportFigure 5.32Cells only export fully processed mRNAFigure 5.327Nuclear ExportHIV Rev protein• viral protein that exports unspliced and partially spliced viral mRNA• Contains a nuclear export signal.• Binds to specific RNA sequence in alternatively spliced introns termed Rev-responsive element (RRE)Problem: cells only export fully processedFig. 10.15 Fig. 10.17Export of viral mRNA by cellular proteinsConstitutive transport elements (CTE): Specific sequence in unsplicedviral mRNAs that promote export (table 10.3)Fig. 10.18Export of Single-Exon mRNAAppendix A Fig 8 &9 Many viral mRNA do not contain introns and therefore are notspliced.Termed single-exon mRNAposttranscriptional regulatory element (PRE)- necessary for efficientexport of viral mRNA.•function in the absence of viral proteins•may utilized cellular export pathwaysExport mediated by viral protein: