Chapter 8: Processing

Processing Introduction Viral messenger RNAs (mRNAs) are synthesized by either viral or cellular enzymes and may be made in the nucleus or the cytoplasm of an infected cell A series of covalent modifications collectively known as RNA processing, facilitate recognition of mRNAs by the protein synthesis machinery and translation of the coding sequences by cellular ribosomes Two modifications important for efficient translation are the addition of m7GpppN to the 5' end (capping) and the addition of multiple A nucleotides to the 3' end (polyadenylation) During splicing, short blocks at noncontiguous coding sequences (exons) are joined precisely to create a complete protein-coding sequence for translation, while the intervening sequences (introns) are discarded Such RNA editing reactions introduce nucleotides that are not encoded in the genome, and consequently may change the sequence of the encoded protein Regulation of RNA processing can increase the coding capacity of the viral genome, determine when specific viral proteins are made during the infectious cycle, and facilitate selective expression of viral genetic information Covalent modification during viral pre-mRNA processing Capping the 5' ends of viral mRNA This structure protects mRNAs from 5' exonucleolytic attack and is essential for the efficient translation of most mRNAs, because it is recognized by translation initiation proteins Although most viral mRNAs carry a 5'-terminal cap, this modification is made by three different mechanisms: de novo synthesis by cellular enzymes, synthesis by viral enzymes, and acquisition of preformed 5' cap structures from cellular pre-mRNAs or mRNAs Synthesis of viral 5' cap structures by cellular enzymes Phosphorylation of paused RNA polymerase II at specific serine residues in the Cterminal domain of the largest subunit is the signal for binding of the capping enzyme and capping of the nascent RNA In mammalian cells, a single protein, commonly called capping enzyme, contains the two activities required for synthesis of a 5' cap Methylation of the guanine base added during capping is important for recognition of mRNA by the translation machinery, whereas 2 -0 methylation of the sugar(s) block the inhibition of translation induced by interferons, major components of the initial antiviral defenses Synthesis of viral 5' cap structures by viral enzymes When viral mRNAs are made in the cytoplasm of infected cells, their 5 cap structures are, of necessity, made by viral enzymes Most viral capping enzymes cooperate with viral RNA-dependent RNA polymerases that synthesize both (-) and (+) strand RNAs, but cap only (+) strand RNAs Acquisition of viral 5' cap structures from cellular RNAs The 5' caps of these viral mRNAs are acquired when viral cap-dependent endonucleases cleave cellular transcripts to produce the primers needed for viral mRNA synthesis, a process called cap snatching Synthesis of 3' poly(A) segments of viral mRNA Those RNAs that are not endowed with a 3' poly(A) tail, such as reoviral and arenaviral mRNAs, may survive by virtue of 3'-terminal stem-loop structures that block nucleolytic attack The addition of 3' poly(A) segments to viral pre-mRNAs, like capping of their 5' ends, can be carried out by either cellular or viral enzymes Polyadenylation of viral pre-mRNA by cellular enzymes Transcription of a viral or cellular gene by RNA polymerase II proceeds beyond the site at which poly(A) will be added The first reaction in polyadenylation is recognition of this sequence by the multimeric protein termed CPSF (cleavage and polyadenylation specificity protein), an interaction that is stabilized by other proteins Like capping, polyadenylation at the 3' ends of an mRNA appears to be coordinated with synthesis of the pre-mRNA: binding of both CPSF and CSTF (cleavage stimulator protein) to the C-terminal domain of the largest subunit of RNA polymerase II is essential for polyadenylation Polyadenylation of viral pre-mRNAs by viral enzymes Synthesis of poly(A) tails by viral enzymes can occur either posttranscriptionally, like polyadenylation of cellular mRNAs, or during viral mRNA synthesis Formation of 3'ends by termination of transcription The 3' ends of vaccinia virus early mRNAs are formed by termination of transcription by the viral DNA-dependent RNA polymerase At specific sites, a mechanism with no known counterpart in cellular mRNA synthesis systems These dual-function viral RNA-processing proteins seem likely to facilitate coordination of the reactions by which viral mRNAs are produced Polyadenylation during viral mRNA synthesis The poly(A) sequences of other mRNAs made by viral RNA polymerases are produced during synthesis of the mRNA, rather than posttranscriptionally The unusual poly(A) tracts that comprise the 5' untranslated regions of vaccinia virus late mRNAs are thought to be synthesized by a similar reiterative copying mechanism Internal methylation of adenosine residues Discovery of internal methylation Methylation of internal A residues proved to be a common modification of eukaryotic mRNAs, and a conserved methyltransferase that catalyzes this modification was identified in the 1990's Stimulation of viral mRNA production and translation When the majority of the 11 sites of m6A methylation mapped subsequently were replaced by synonymous mutations, viral late mRNA export was reduced Similarly, mutations that eliminate sites of m6A methylation from the (-) and (+) strands of the influenza virus HA genome segment led to reduced synthesis of HA mRNA but an even greater decrease in production of the HA protein Inhibition of virus reproduction Rather, cytoplasmic proteins that recognize internal m6A bind to specific residues modified in this way in the viral genome to impair its association with the viral core protein and lipid droplets, the sites of assembly, and hence release of virus particles Splicing of viral pre-mRNA Discovery of splicing Such hnRNAs (heterogeneous nuclear RNAs) were shown to carry both 5'-terminal cap structures and 3' poly(A) sequences, leading to the conclusion that both ends of the hnRNA were preserved in the smaller, mature mRNA The demonstration that primary major late transcripts contain the introns confirmed that mature mRNAs are formed by splicing of noncontiguous coding sequences in the pre-mRNA Splicing occurs in all eukaryotic cells, and the great majority of mammalian premRNAs (94%), like the adenoviral major late mRNAs, comprise exons separated by introns Introns are generally much longer than exons, and only short sequences at their ends are necessary for accurate splicing Consequently, introns provide numerous sites at which DNA sequences can be broken and rejoined without loss of coding information, and greatly increase the frequency with which random recombination reactions can create new functional genes by rearrangement of exons Mechanism of splicing Sequencing of DNA copies of a large number of cellular and viral mRNAs and of the genes that encode them identified short consensus sequences at the 5' and 3' splice sites, which are joined to each other in mature mRNA Pre-mRNA splicing occurs by two transesterification reactions, in which one phosphodiester bond is exchanged for another without the need for an external supply of energy However, each splicing reaction must be completed with a high degree of accuracy to ensure that coding information is not lost or altered, and the chemically active hydroxyl groups must also be brought into close proximity to the phosphodiester bonds they will attack Splicing comprises assembly and activation of a complete spliceosome, catalysis of the reactions, and disassembly and release of spliced mRNA It has long been suspected that the spliceosome might be an RNA enzyme (or ribozyme), and compelling evidence for catalysis by U6 snRNAs during splicing has been reported Splicing of pre-mRNAs is commonly cotranscriptional, and components of the splicing machinery associate with the hyperphosphorylated form of the Cterminal domain of the largest subunit of RNA polymerase II during elongation of transcription Alternative splicing However, numerous cellular and many viral pre-mRNAs yield more than one mRNA as a result of the splicing of different combinations of exons, a process termed alternative splicing Alternative splicing can also be important Alternative splicing for temporal regulation of viral gene expression, or the control of a critical Such splicing, which comprise balance in the production of spliced and selection among large numbers unspliced mRNAs of exons in a pre-mRNA, is Regulated processing of viral pre-mRNA governed by multiple Cellular differentiation regulates production of parameters, including splice papillomaviral late pre-mRNAs site and regulatory sequences The late stages of the infectious cycles of in the pre-mRNA and the mammalian papillomaviruses, including constellation of splicing genome amplification, synthesis of late proteins present in particular proteins, and assembly and release of cell types progeny virus particles, are restricted to highly differentiated keratinocytes in the outer layers of an epithelium Production of the late mRNAs requires utilization of both polyadenylation and splice sites that are not recognized in undifferentiated cells, and suppression of recognition of the polyadenylation site for early mRNAs Differentiation-dependent alterations in concentration of cellular RNA-binding proteins also govern the efficiency of recognition of the early poly(A) addition Production of spliced and unspliced RNAs essential for virus reproduction Features that maintain the proper splicing balance include suboptimal recognition of the 3' splice site, and a splicing enhancer in the adjacent exon Nevertheless, alternative splicing is also regulated by specific sequences that promote or repress recognition or utilization of splice sites and by the degree of conformity of 3' splice sites to the optimal sequence Sequences present only at the 3' end of the pre-mRNA facilitate binding of CPSF to the nearby 5 AAUAAA3 sequence to stimulate polyadenylation in vitro and in infected cells in culture Temporal regulation of synthesis of adenoviral major late mRNAs The production of adenoviral major late mRNAs epitomizes complex alternative splicing and polyadenylation at multiple sites in a pre-mRNA, which in this case gives rise to at least 15 different mRNAs High-efficiency polyadenylation at the L1 site during the early phase of infection prevents synthesis of L2 to L5 mRNAs It is therefore likely that these poly (A) addition signals compete more effectively with the L1 site for components of the polyadenylation machinery later in the infectious cycle Editing of viral mRNAs Viral mRNAs are edited by either insertion of nucleotides not directly specified in the template during synthesis or alteration of a base in site, changing the sequence and function of the protein specified by the edited mRNA Editing during mRNA synthesis During the mRNA synthesis, the viral RNA polymerase inserts one or two G residues at specific positions in a fraction of the RNA molecules Similarly, RNA editing determines whether an Ebola virus gene is expressed as a full-length glycoprotein that is required for virus entry, or as a truncated secreted protein, and modulates the cytotoxicity of the virus Editing following mRNA synthesis Editing after synthesis of mRNAs can be accomplished by cellular enzymes that dominate adenosine bases in double-stranded RNA regions to form inosine (I) The large delta antigen, which is necessary for particle assembly and inhibits replication of the RNA genome, is made when editing converts a UAG termination codon to the UGG (tryptophan) codon This modification appears to regulate the function of the kaposin protein: this protein exhibits transforming activity, but only when it is made from the unedited coding sequence and editing of this site predominates in productively infected cells Export of RNAs from the nucleus Any mRNA made in the nucleus must be transported to the cytoplasm for translation. Other classes of RNA, including small cellular and viral RNAs made by RNA polymerase III, also enter the cytoplasm permanently or transiently The cellular export machinery Like proteins entering the nucleus, RNA molecules travel between nuclear and cytoplasmic compartments via the nuclear pore complexes Export of RNA molecules also shares several mechanistic features with import of proteins into the nuclei: substrates for nuclear export or import are identified by specific protein signals, and some soluble proteins, including the small guanosine nucleotide-binding protein RAN, function in both import and export Export of viral mRNA All viral mRNAs made in infected cell nuclei carry the same 5 - and 3'-terminal modifications as cellular mRNAs that are exported In addition, a protein complex that marks mature mRNAs for export is assembled on the RNA only during splicing, and efficient export requires cooperation among multiple adapter proteins that are deposited as a pre-mRNA is processed The human immunodeficiency virus type I Rev protein directs export of intron containing mRNAs Rev binds specifically to an RNA sequence termed the Rev-responsive element that lies within an alternatively spliced intron of viral pre-mRNA Export of RNAs that contain the Rev-responsive element depends on the formation of these RNA bound oligomers, and a leucine rich nuclear export signal present in Rev Translocation of the complex containing viral RNAs, Rev, and cellular proteins through the nuclear pore complex requires special nucleoporins and other poring The human immunodeficiency virus type I Rev protein therefore circumvents the normal restriction on the export of intron-containing pre-mRNAs from the host cell nucleus by diverting such viral mRNAs to a cellular pathway that handles intron-less RNAs Export of unspliced viral mRNAs The majority [unspliced RNA] encode regulatory proteins, such as components of signal transduction pathways and cytokines Posttranscriptional regulation of viral or cellular gene expression by viral proteins Temporal control of viral gene expression Regulation of alternative splicing and polyadenylation by viral proteins The regulation of alternative polyadenylation or alternative splicing by viral proteins can be critical for changes in patterns of viral gene expression as the infections cycle progresses Entry into the late phase of adenovirus infection is also marked by changes in the ratios of alternatively spliced mRNA products of several viral pre-mRNAs The viral Tat protein is necessary for synthesis of full-length transcripts of proviral DNA, but when modified by acetylation also sequesters a kinase (CDK13) that phosphorylates SF2 The viral NP1 protein suppresses recognition of this poly(A) addition site and promotes splicing to remove the sequence immediately upstream, favoring production of mRNAs specifying the capsid proteins Regulation of mRNA export Even though all are encoded within a single proviral transcription unit, the regulatory and structural proteins of human immunodeficiency virus type I are made sequentially in infected cells, as a result of regulation of mRNA export by the Rev protein: This protein effects a switch in viral gene expression from early production of viral regulatory proteins to a later phase, in which components of virus particles are made Viral proteins can inhibit cellular mRNA production Inhibition of polyadenylation and splicing When NS1 is not made, infected cells produce larger quantities of cellular mRNAs that encode interferons and other proteins with antiviral activities Inhibition of cellular mRNA export Facilitating production of viral mRNAs Adenovirus infection induces severe inhibition of cellular protein synthesis during the late phase of infection When selective viral mRNA export is prevented by mutations in the viral genome, both the quantities of late proteins made in infected cells and virus yield are decreased substantially In this case, binding of the viral protein to the cellular export proteins both impairs export from the nucleus of a subset of cellular mRNAs and promotes efficient expression of viral late genes Impairing antiviral responses Inhibition of export of cellular RNAs from the nucleus can reduce competition of cellular with viral mRNAs for components of the translational machinery The consequent disruption of cellular mRNA export probably reduces host cell protein synthesis and also appears to block the important antiviral defense mediated by type I interferons, potent antiviral cytokines Regulation of turnover of viral and cellular mRNAs in the cytoplasm Intrinsic turnover Individual mRNAs may differ in the rate at which they are translated, and also in such properties as cytoplasmic location and stability This property is described in terms of the time required for 50% of the mRNA population to be degraded under conditions in which replenishment of the cytoplasmic pool is blocked, the half-life of the mRNA The stabilities of viral mRNAs have not been examined in much detail, in part because many viral infectious cycles are completed within the normal range of mRNA half-lives and many viral mRNAs carry the 5' caps and 3' poly(A) tails that protect against degradation ZAP proteins bind to specific sequences in cytoplasmic retroviral RNAs and have been reported to recruit cellular enzymes that catalyze deadenylation and decaying Regulation of mRNA stability by viral proteins Vhs is a structural protein: it is present at low concentrations in the regiment and hence delivered to infected cells at the start of the infectious cycle Although more Vhs protein is made in infected cells once its coding sequence is expressed during the late phase of infection, the protein is sequestered in the tegument of assembling virus particles It has been suggested that turnover of viral mRNAs may facilitate the production of specific sets of viral proteins during the successive phases of the infectious cycle Nonsense-mediated mRNA decay Such nonsense-mediated decay prevents synthesis of truncated proteins that could be detrimental to cellular physiology, for example, because they function as dominant negative inhibitors, and serves a broader control function In one pathway, splicing is coupled to translational quality control by deposition of the multimeric exon junction complex (EJC) as exons in the mRNA are spliced. The second mechanism is triggered by long 3' untranslated regions The genomes of (+) strand RNA viruses that also serve as mRNAs often contain multiple open reading frames and hence internal termination codons and long 3' untranslated regions, features that would be expected to trigger nonsense-mediated decay The capsid proteins of this and other flaviviruses have also been reported to interact with a component of the EJC to increase accumulation of viral RNA genomes in infecting cells by inhibiting nonsense-mediated decay Noncoding RNAs Small interfering RNAs and micro-RNAs Discovery and synthesis RNA interference is mediated by small RNA molecules (typically 19 to 25 nucleotides in length) that function in antiviral defense or regulate gene expression Small interfering RNAs (siRNAs), such as those first discovered in plants, are initially processed by endonucleolytic cleavage of double-stranded RNAs in the cytoplasm by dicer enzymes The sequences that encode miRNAs are often clustered, an arrangement that allows synthesis of transcripts containing multiple miRNA sequences These RNAs are then unwound from one 5' end, and one strand becomes tightly associated with a member of the argonaut (AGO) family of proteins in the effector ribonucleoprotein, termed the RNA induced silencing complex (RISC) Cellular miRNAs in virus-infected cells Cellular miRNA-155 promotes viral oncogenesis This miRNA [miRNA-155], which is present at high concentrations in human B-cell lymphomas and tumors, is encoded by a gene first identified as a common integration site of the retrovirus avian leukosis virus miRNA-155 promotes B-cell proliferation by blocking a signaling pathway that activates the tumor suppressor RB, but whether this mechanism contributes to Epstein-Barr virus oncogenesis is not yet known Viral micro-RNAs A retroviral miRNA that fosters oncogenesis Although viral mRNAs are present At only low concentrations in persistently infected B cells in vivo, viral miRNAs accumulate massively, as they do in tumors, suggesting that these miRNAs might contribute to oncogenesis Long noncoding RNAs Cellular lncRNAs are classified according to the positions of their coding sequences relative to neighboring protein-coding genes as antisense, bidirectional, long intergenic, intronic, and enhancer lncRNAs