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.

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.

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.

A Conserved Splicing Silencer Dynamically Regulates O-GlcNAc Transferase Intron Retention and O-GlcNAc Homeostasis.

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS) that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.

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.

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.

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.

Viral factors reveal a role for REF/Aly in nuclear RNA stability.

TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In Saccharomyces cerevisiae, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV noncoding polyadenylated nuclear (PAN) RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation, and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independently of their export and support a broader conservation of RNA quality control mechanisms from yeast to humans.