The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor.

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

Functional analysis of 3'-UTR hairpins supports a two-tiered model for posttranscriptional regulation of MAT2A by METTL16.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells need to carefully control SAM levels. MAT2A encodes the only SAM synthetase expressed in the majority of human cells, and its 3'-UTR has six conserved regulatory hairpins (hp1-6) that can be methylated by the N6-methyladenosine methyltransferase METTL16. Hp1 begins 8 nt from the stop codon, whereas hp2-6 are clustered further downstream (∼800 nt). These hairpins have been proposed to regulate MAT2A mRNA levels in response to intracellular SAM levels by regulating intron detention of the last intron of MAT2A and by modulating the stability of the fully spliced mRNA. However, a dissection of these two posttranscriptional mechanisms has not been previously reported. Using a modular reporter system, we show that hp1 functions primarily when the detained intron is included in the reporter and when that intron has a suboptimal polypyrimidine tract. In contrast, the hp2-6 cluster modulates mRNA stability independent of the detained intron, although hp1 may make a minor contribution to the regulation of decay as well. Taken with previously published reports, these data support a two-tiered model for MAT2A posttranscriptional regulation by METTL16 through its interactions with hp1 and hp2-6. In the upstream tier, hp1 and METTL16 control MAT2A intron detention, whereas the second tier involves METTL16-dependent methylation of hp2-6 to control MAT2A mRNA stability. Thus, cells use a similar set of molecular factors to achieve considerable complexity in the posttranscriptional regulation of SAM homeostasis.

Kaposi's Sarcoma-Associated Herpesvirus Fine-Tunes the Temporal Expression of Late Genes by Manipulating a Host RNA Quality Control Pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic nuclear DNA virus that expresses its genes using the host cell transcription and RNA processing machinery. As a result, KSHV transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, the PABPN1- and poly(A) polymerase α/γ (PAPα/γ)-mediated RNA decay (PPD) pathway and an ARS2-dependent decay pathway. Here, we present global analyses of viral transcript levels to further understand the roles of these decay pathways in KSHV gene expression. Consistent with our recent report that the KSHV ORF57 protein increases viral transcript stability by impeding ARS2-dependent decay, ARS2 knockdown has only modest effects on viral gene expression 24 h after lytic reactivation of wild-type virus. In contrast, inactivation of PPD has more widespread effects, including premature accumulation of late transcripts. The upregulation of late transcripts does not require the primary late-gene-specific viral transactivation factor, suggesting that cryptic transcription produces the transcripts that then succumb to PPD. Remarkably, PPD inactivation has no effect on late transcripts at their proper time of expression. We show that this time-dependent PPD evasion by late transcripts requires the host factor nuclear RNAi-defective 2 (NRDE2), which has previously been reported to protect cellular RNAs by sequestering decay factors. From these studies, we conclude that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi's sarcoma and other lymphoproliferative disorders. Nuclear expression of KSHV genes results in exposure to at least two host-mediated nuclear RNA decay pathways, the PABPN1- and PAPα/γ-mediated RNA decay (PPD) pathway and an ARS2-mediated decay pathway. Perhaps unsurprisingly, we previously found that KSHV uses specific mechanisms to protect its transcripts from ARS2-mediated decay. In contrast, here we show that PPD is required to dampen the expression of viral late transcripts that are prematurely transcribed, presumably due to cryptic transcription early in infection. At the proper time for their expression, KSHV late transcripts evade PPD through the activity of the host factor NRDE2. We conclude that KSHV fine-tunes the temporal expression of its genes by modulating PPD activity. Thus, the virus both protects from and exploits the host nuclear RNA decay machinery for proper expression of its genes.

Kaposi's sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment.

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

Balance between MAT2A intron detention and splicing is determined cotranscriptionally.

Transcriptome analysis of human cells has revealed that intron retention controls the expression of a large number of genes with diverse cellular functions. Detained introns (DI) constitute a subgroup of transcripts with retained introns that are not exported to the cytoplasm but instead remain in the nucleus. Previous studies reported that the splicing of DIs in the CLK1 transcript is post-transcriptionally induced to produce mature mRNA in the absence of new transcription. Thus, CLK1-DI serves as a precursor or "reservoir" for the CLK1 mRNA. However, whether this is a universal mechanism for gene regulation by intron detention remains unknown. The MAT2A gene encodes S-adenosylmethionine (SAM) synthetase and it contains a DI that is regulated in response to intracellular SAM levels. We used three independent assays to assess the precursor-product relationship between MAT2A-DI and MAT2A mRNA. In contrast to CLK1-DI, these data support a model in which the MAT2A-DI transcript is not a precursor to mRNA but is instead a "dead-end" RNA fated for nuclear decay. Additionally, we show that in SAM-deprived conditions the cotranscriptional splicing of MAT2A detained introns increases. We conclude that polyadenylated RNAs with DIs can have at least two distinct fates. They can serve as nuclear reservoirs of pre-mRNAs available for rapid induction by the cell, or they constitute dead-end RNAs that are degraded in the nucleus.

Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting.

The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

ADAR2 regulates RNA stability by modifying access of decay-promoting RNA-binding proteins.

Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3΄UTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

Canonical Poly(A) Polymerase Activity Promotes the Decay of a Wide Variety of Mammalian Nuclear RNAs.

The human nuclear poly(A)-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs). In addition, PABPN1 promotes hyperadenylation by stimulating poly(A)-polymerases (PAPα/γ), but this activity has not previously been linked to the decay of endogenous transcripts. Moreover, the mechanisms underlying target specificity have remained elusive. Here, we inactivated PAP-dependent hyperadenylation in cells by two independent mechanisms and used an RNA-seq approach to identify endogenous targets. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and promoter upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD). Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. Additional investigation showed that a genetically-encoded poly(A) tail is sufficient to drive decay, suggesting that degradation occurs independently of the canonical cleavage and polyadenylation reaction. Surprisingly, treatment with transcription inhibitors uncouples polyadenylation from decay, leading to runaway hyperadenylation of nuclear decay targets. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts.

HITS-CLIP analysis uncovers a link between the Kaposi's sarcoma-associated herpesvirus ORF57 protein and host pre-mRNA metabolism.

The Kaposi's sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5' ends. The position of these 5'-bound fragments correlated closely with the 5'-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.

Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy.

Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency.

The human nuclear poly(a)-binding protein promotes RNA hyperadenylation and decay.

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

Chromatin immunoprecipitation and microarray analysis suggest functional cooperation between Kaposi's Sarcoma-associated herpesvirus ORF57 and K-bZIP.

The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 57 (ORF57)-encoded protein (Mta) is a multifunctional regulator of viral gene expression. ORF57 is essential for viral replication, so elucidation of its molecular mechanisms is important for understanding KSHV infection. ORF57 has been implicated in nearly every aspect of viral gene expression, including transcription, RNA stability, splicing, export, and translation. Here we demonstrate that ORF57 interacts with the KSHV K-bZIP protein in vitro and in cell extracts from lytically reactivated infected cells. To further test the biological relevance of the interaction, we performed a chromatin immunoprecipitation and microarray (ChIP-chip) analysis using anti-ORF57 antibodies and a KSHV tiling array. The results revealed four specific areas of enrichment, including the ORF4 and K8 (K-bZIP) promoters, as well as oriLyt, all of which interact with K-bZIP. In addition, ORF57 associated with DNA corresponding to the PAN RNA transcribed region, a known posttranscriptional target of ORF57. All of the peaks were RNase insensitive, demonstrating that ORF57 association with the viral genome is unlikely to be mediated exclusively by an RNA tether. Our data demonstrate that ORF57 associates with the viral genome by using at least two modes of recruitment, and they suggest that ORF57 and K-bZIP coregulate viral gene expression during lytic infection.

Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics.

A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N(6) pA can be visualized and purified by using click chemistry.

Kaposi's sarcoma-associated herpesvirus ORF57 protein binds and protects a nuclear noncoding RNA from cellular RNA decay pathways.

The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless beta-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway.

Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention.

Intron retention (IR) regulates gene expression to control fundamental biological processes like metabolism, differentiation, and cell cycle. Despite a wide variety of genes controlled by IR, few techniques are available to identify regulators of IR in an unbiased manner. Here, we describe a CRISPR knockout screening method that can be applied to uncover regulators of IR. This method uses GFP reporter constructs containing a retained intron from a gene of interest such that GFP signal is regulated by IR in the same fashion as the endogenous gene. The GFP levels are then used as a readout for genome-wide CRISPR screening. We have successfully used this approach to identify novel regulator of IR of the MAT2A transcript and propose that similar screens will be broadly applicable for the identification of novel factors that control IR of specific transcripts.

The PNUTS-PP1 complex acts as an intrinsic barrier to herpesvirus KSHV gene expression and replication.

Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-PP1 complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion enhances global KSHV gene expression and overall viral replication. Mechanistically, PNUTS requires PP1 interaction, binds viral RNAs downstream of polyadenylation sites, and restricts transcription readthrough of viral genes. Surprisingly, PNUTS also represses productive elongation at the 5´ ends of the KSHV reporter and the KSHV T1.4 RNA. From these data, we conclude that PNUTS' activity constitutes an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.

Posttranscriptional gene regulation in Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some cases of multicentric Castleman's disease. To understand the pathogenesis and life cycle of KSHV, significant focus has been placed on determining how KSHV factors influence viral and cellular gene expression. The importance of transcriptional regulation by KSHV is well documented, but several KSHV posttranscriptional regulators are also essential for KSHV replication and pathogenesis. KSHV miRNAs regulate translation and stability of cellular mRNAs that may be important for tumorigenesis. The ORF57 protein has been reported to enhance several posttranscriptional processes including viral mRNA export, RNA stability and pre-mRNA splicing. SOX, Kaposin B and the PAN-ENE regulate the stability of viral or cellular transcripts. Together, these observations point to the importance of posttranscriptional regulation in KSHV. With the growing appreciation of posttranscriptional regulation in cellular gene expression, it seems likely that the list of viral posttranscriptional regulatory schemes will expand as new details of KSHV gene regulation are uncovered.

Poly(A) tail length regulates PABPC1 expression to tune translation in the heart.

The rate of protein synthesis in the adult heart is one of the lowest in mammalian tissues, but it increases substantially in response to stress and hypertrophic stimuli through largely obscure mechanisms. Here, we demonstrate that regulated expression of cytosolic poly(A)-binding protein 1 (PABPC1) modulates protein synthetic capacity of the mammalian heart. We uncover a poly(A) tail-based regulatory mechanism that dynamically controls PABPC1 protein synthesis in cardiomyocytes and thereby titrates cellular translation in response to developmental and hypertrophic cues. Our findings identify PABPC1 as a direct regulator of cardiac hypertrophy and define a new paradigm of gene regulation in the heart, where controlled changes in poly(A) tail length influence mRNA translation.