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Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes., (© 2016 Marayati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Most mammalian protein coding genes are subject to alternative cleavage and polyadenylation (APA), which can generate distinct mRNA 3'UTRs with differing regulatory potential. Although this process has been intensely studied in recent years, it remains unclear how and to what extent cleavage site selection is regulated under different physiological conditions. The cleavage factor Im (CFIm) complex is a core component of the mammalian cleavage machinery, and the observation that its depletion causes transcriptome-wide changes in cleavage site use makes it a key candidate regulator of APA. This review aims to summarize current knowledge of the CFIm complex, and explores the evidence surrounding its potential contribution to regulation of APA., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

In eukaryotes, bulk histone expression occurs in the S phase of the cell cycle. This highly conserved system is crucial for genomic stability and proper gene expression. In metazoans, Stem-loop binding protein (SLBP), which binds to 3' ends of canonical histone mRNAs, is a key factor in histone biosynthesis. SLBP is mainly expressed in S phase and this is a major mechanism to limit bulk histone production to the S phase. At the end of S phase, SLBP is rapidly degraded by proteasome, depending on two phosphorylations on Thr 60 and Thr 61. Previously, we showed that SLBP fragment (aa 51-108) fused to GST, is sufficient to mimic the late S phase (S/G2) degradation of SLBP. Here, using this fusion protein as bait, we performed pull-down experiments and found that DCAF11, which is a substrate receptor of CRL4 complexes, binds to the phosphorylated SLBP fragment. We further confirmed the interaction of full-length SLBP with DCAF11 and Cul4A by co-immunoprecipitation experiments. We also showed that DCAF11 cannot bind to the Thr61/Ala mutant SLBP, which is not degraded at the end of S phase. Using ectopic expression and siRNA experiments, we demonstrated that SLBP expression is inversely correlated with DCAF11 levels, consistent with the model that DCAF11 mediates SLBP degradation. Finally, we found that ectopic expression of the S/G2 stable mutant SLBP (Thr61/Ala) is significantly more toxic to the cells, in comparison to wild type SLBP. Overall, we concluded that CRL4-DCAF11 mediates the degradation of SLBP at the end of S phase and this degradation is essential for the viability of cells.

A rare subset of HIV-1-infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1-infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1-infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection.

The nontemplated addition of single or multiple nucleotides to RNA transcripts is an efficient means to control RNA stability and processing. Cytoplasmic RNA adenylation and the less well-known uridylation are post-transcriptional mechanisms regulating RNA maturation, activity, and degradation. Gld2 is a member of the noncanonical poly(A) polymerases, which include enzymes with varying nucleotide specificity, ranging from strictly ATP to ambiguous to exclusive UTP adding enzymes. Human Gld2 has been associated with transcript stabilizing miRNA monoadenylation and cytoplasmic mRNA polyadenylation. Most recent data have uncovered an unexpected miRNA uridylation activity, which promotes miRNA maturation. These conflicting data raise the question of Gld2 nucleotide specificity. Here, we biochemically characterized human Gld2 and demonstrated that it is a bona fide adenylyltransferase with only weak activity toward other nucleotides. Despite its sequence similarity with uridylyltransferases (TUT4, TUT7), Gld2 displays an 83-fold preference of ATP over UTP. Gld2 is a promiscuous enzyme, with activity toward miRNA, pre-miRNA, and polyadenylated RNA substrates. Apo-Gld2 activity is restricted to adding single nucleotides and processivity likely relies on additional RNA-binding proteins. A phylogeny of the PAP/TUTase superfamily suggests that uridylyltransferases, which are derived from distinct adenylyltransferase ancestors, arose multiple times during evolution via insertion of an active site histidine. A corresponding histidine insertion into the Gld2 active site alters substrate specificity from ATP to UTP., (© 2016 Chung et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Unlabelled: Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4(+) T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains., Importance: CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

Human immunodeficiency virus (HIV) patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), have been known to develop neuropathic pain. While there has been a major shift away from some neurotoxic NRTIs in current antiretroviral therapy, a large number of HIV patients alive today have previously received them, and many have developed painful peripheral neuropathy. The exact mechanisms by which HIV with NRTIs contribute to the development of neuropathic pain are not known. Previous studies suggest that cytoplasmic polyadenylation element-binding protein (CPEB), reactive oxygen species (ROS), and cAMP-response element-binding protein (CREB)-binding protein (CBP), are involved in the neuroimmunological diseases including inflammatory/neuropathic pain. In this study, we investigated the role of CPEB, mitochondrial ROS (mtROS), or CBP in neuropathic pain induced by HIV envelope protein gp120 combined with antiretroviral drug. The application of recombinant gp120 into the sciatic nerve plus systemic ddC (one of NRTIs) induced mechanical allodynia. Knockdown of CPEB or CBP using intrathecal antisense oligodeoxynucleotide (AS-ODN) reduced mechanical allodynia. Intrathecal mitochondrial superoxide scavenger mito-tempol (Mito-T) increased mechanical withdrawal threshold. Knockdown of CPEB using intrathecal AS-ODN, reduced the up-regulated mitochondrial superoxide in the spinal dorsal horn in rats with gp120 combined with ddC. Intrathecal Mito-T lowered the increased expression of CBP in the spinal dorsal horn. Immunostaining studies showed that neuronal CPEB positive cells were co-localized with MitoSox positive profiles, and that MitoSox positive profiles were co-localized with neuronal CBP. Our studies suggest that neuronal CPEB-mtROS-CBP pathway in the spinal dorsal horn, plays an important role in the gp120/ddC-induced neuropathic pain in rats., (Copyright © 2016. Published by Elsevier Inc.)

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m(6)A-modified RNA., (© 2016 Chatterjee et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3' end by 3'-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3'-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3' end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation.

In mouse mammary epithelial cells, cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the apical localization of ZO-1 mRNA, which encodes a critical tight junction component. In mice lacking CPEB1 and in cultured cells from which CPEB has been depleted, randomly distributed ZO-1 mRNA leads to the loss of cell polarity. We have investigated whether this diminution of polarity results in an epithelial-to-mesenchyme (EMT) transition and possible increased metastatic potential. Here, we show that CPEB1-depleted mammary epithelial cells alter their gene expression profile in a manner consistent with an EMT and also become motile, which are made particularly robust when cells are treated with transforming growth factor-β, an enhancer of EMT. CPEB1-depleted mammary cells become metastatic to the lung following injection into mouse fat pads while ectopically expressed CPEB1 prevents metastasis. Surprisingly, CPEB1 depletion causes some EMT/metastasis-related mRNAs to have shorter poly(A) tails while other mRNAs to have longer poly(A) tails. Matrix metalloproteinase 9 (MMP9) mRNA, which encodes a metastasis-promoting factor, undergoes poly(A) lengthening and enhanced translation upon CPEB reduction. Moreover, in human breast cancer cells that become progressively more metastatic, CPEB1 is reduced while MMP9 becomes more abundant. These data suggest that at least in part, CPEB1 regulation of MMP9 mRNA expression mediates metastasis of breast cancer cells.

Spliceosome and 3'-end processing complexes are necessary for the precursor mRNA (pre-mRNA) maturation. Spliceosome complex removes noncoding introns, while 3'-end processing involves in cleavage and addition of poly(A) tails to the nascent transcript. Rna14 protein in budding yeast has been implicated in cleavage and polyadenylation of mRNA in the nucleus but their role in the pre-mRNA splicing has not been studied. Here, we report the isolation of a mutant allele of rna14 in fission yeast, Schizosaccharomyces pombe that exhibits reduction in protein level of Chk1 at the nonpermissive temperature, primarily due to the defects in posttranscriptional processing. Reverse transcriptase-polymerase chain reaction analysis reveals defective splicing of the chk1(+) transcript at the nonpermissive temperature. Apart from chk1(+), the splicing of some other genes were also found to be defective at the nonpermissive temperature suggesting that Rna14 might be involved in pre-mRNA splicing. Subsequently, genetic interaction of Rna14 with prp1 and physical interactions with Prp28 suggest that the Rna14 might be part of a larger protein complex responsible for the pre-mRNA maturation.

RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. In vitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(β,γ-methylene)triphosphate (AMP-PCP) is an effective in vitro 3' cleavage inhibitor with an IC50 of ∼300 μM, but that most other ATP analogs, including adenosine 5'-(β,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation in vitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage., Competing Interests: We confirm that there is no conflict of interest., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Unlabelled: The viral capsid of HIV-1 interacts with a number of host factors to orchestrate uncoating and regulate downstream events, such as reverse transcription, nuclear entry, and integration site targeting. PF-3450074 (PF74), an HIV-1 capsid-targeting low-molecular-weight antiviral compound, directly binds to the capsid (CA) protein at a site also utilized by host cell proteins CPSF6 and NUP153. Here, we found that the dose-response curve of PF74 is triphasic, consisting of a plateau and two inhibitory phases of different slope values, consistent with a bimodal mechanism of drug action. High PF74 concentrations yielded a steep curve with the highest slope value among different classes of known antiretrovirals, suggesting a dose-dependent, cooperative mechanism of action. CA interactions with both CPSF6 and cyclophilin A (CypA) were essential for the unique dose-response curve. A shift of the steep curve at lower drug concentrations upon blocking the CA-CypA interaction suggests a protective role for CypA against high concentrations of PF74. These findings, highlighting the unique characteristics of PF74, provide a model in which its multimodal mechanism of action of both noncooperative and cooperative inhibition by PF74 is regulated by interactions of cellular proteins with incoming viral capsids., Importance: PF74, a novel capsid-targeting antiviral against HIV-1, shares its binding site in the viral capsid protein (CA) with the host factors CPSF6 and NUP153. This work reveals that the dose-response curve of PF74 consists of two distinct inhibitory phases that are differentially regulated by CA-interacting host proteins. PF74's potency depended on these CA-binding factors at low doses. In contrast, the antiviral activity of high PF74 concentrations was attenuated by cyclophilin A. These observations provide novel insights into both the mechanism of action of PF74 and the roles of host factors during the early steps of HIV-1 infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background & Aims: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease., Methods: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed., Results: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation., Conclusions: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Meiotic progression requires exquisitely coordinated translation of maternal messenger (m)RNA that has accumulated during oocyte growth. A major regulator of this program is the cytoplasmic polyadenylation element binding protein 1 (CPEB1). However, the temporal pattern of translation at different meiotic stages indicates the function of additional RNA binding proteins (RBPs). Here, we report that deleted in azoospermia-like (DAZL) cooperates with CPEB1 to regulate maternal mRNA translation. Using a strategy that monitors ribosome loading onto endogenous mRNAs and a prototypic translation target, we show that ribosome loading is induced in a DAZL- and CPEB1-dependent manner, as the oocyte reenters meiosis. Depletion of the two RBPs from oocytes and mutagenesis of the 3' untranslated regions (UTRs) demonstrate that both RBPs interact with the Tex19.1 3' UTR and cooperate in translation activation of this mRNA. We observed a synergism between DAZL and cytoplasmic polyadenylation elements (CPEs) in the translation pattern of maternal mRNAs when using a genome-wide analysis. Mechanistically, the number of DAZL proteins loaded onto the mRNA and the characteristics of the CPE might define the degree of cooperation between the two RBPs in activating translation and meiotic progression., (© 2016. Published by The Company of Biologists Ltd.)

Erh1, the fission yeast homolog of Enhancer of rudimentary, is implicated in meiotic mRNA elimination during vegetative growth, but its function is poorly understood. We show that Erh1 and the RNA-binding protein Mmi1 form a stoichiometric complex, called the Erh1-Mmi1 complex (EMC), to promote meiotic mRNA decay and facultative heterochromatin assembly. To perform these functions, EMC associates with two distinct complexes, Mtl1-Red1 core (MTREC) and CCR4-NOT. Whereas MTREC facilitates assembly of heterochromatin islands coating meiotic genes silenced by the nuclear exosome, CCR4-NOT promotes RNAi-dependent heterochromatin domain (HOOD) formation at EMC-target loci. CCR4-NOT also assembles HOODs at retrotransposons and regulated genes containing cryptic introns. We find that CCR4-NOT facilitates HOOD assembly through its association with the conserved Pir2/ARS2 protein, and also maintains rDNA integrity and silencing by promoting heterochromatin formation. Our results reveal connections among Erh1, CCR4-NOT, Pir2/ARS2, and RNAi, which target heterochromatin to regulate gene expression and protect genome integrity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epileptogenesis in the temporal lobe elicits regulation of gene expression and protein translation, leading to reorganization of neuronal networks. In this process, miRNAs were described as being regulated in a cell-specific manner, although mechanistics of miRNAs activity are poorly understood. The specificity of miRNAs on their target genes depends on their intracellular concentration, reflecting the balance of biosynthesis and degradation. Herein, we confirmed that pilocarpine application promptly (<30 min) induces status epilepticus (SE) as revealed by changes in rat electrocorticogram particularly in fast-beta range (21-30 Hz). SE simultaneously upregulated XRN2 and downregulated PAPD4 gene expression in the hippocampus, two genes related to miRNA degradation and stability, respectively. Moreover, SE decreased the number of XRN2-positive cells in the hilus, while reduced the number of PAPD4-positive cells in CA1. XRN2 and PAPD4 levels did not change in calretinin- and CamKII-positive cells, although it was possible to determine that PAPD4, but not XRN2, was upregulated in parvalbumin-positive cells, revealing that SE induction unbalances the accumulation of these functional-opposed proteins in inhibitory interneurons that directly innervate distinct domains of pyramidal cells. Therefore, we were able to disclose a possible mechanism underlying the differential regulation of miRNAs in specific neurons during epileptogenesis.

Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3'UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3'UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.

Faithful segregation of chromosomes is essential for the maintenance of genome integrity. In a genetic screen to identify genes related to checkpoint function, we have characterized the role of rna14, an essential gene in the maintenance of chromosome dynamics. We demonstrate that Rna14 localizes in the nucleus and in the absence of functional Rna14, the cells exhibit chromosomal segregation defects. The mutant allele of rna14 exhibits genetic interaction with key kinetochore components and spindle checkpoint proteins. Inactivation of rna14 leads to accumulation of Bub1-GFP foci, a protein required for spindle checkpoint activation that could be due to the defects in the attachment of mitotic spindle to the chromosome. Consistently, the double mutant of rna14-11 and bub1 knockout exhibits high degree of chromosome mis-segregation. At restrictive condition, the rna14-11 mutant cells exhibit defects in cell cycle progression with high level of septation. The orthologs of Rna14 in Saccharomyces cerevisiae (sc Rna14) and human (CstF3) contain similar domain architecture and are required for 3'-end processing of pre-mRNA. We have also demonstrated that the fission yeast Rna14 is required to prevent transcriptional read-through. These findings reveal the importance of transcription termination in the maintenance of genomic stability through the regulation of kinetochore function., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The YTH domain-containing protein Mmi1, together with other factors, constitutes the machinery used to selectively remove meiosis-specific mRNA during the vegetative growth of fission yeast. Mmi1 directs meiotic mRNAs to the nuclear exosome for degradation by recognizing their DSR (determinant of selective removal) motif. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif, demonstrating that the Mmi1 YTH domain selectively recognizes the DSR motif. Intriguingly, Mmi1 also contains a potential m(6)A (N(6)-methyladenine)-binding pocket, but its binding of the DSR motif is dependent on a long groove opposite the m(6)A pocket. The DSR-binding mode is distinct from the m(6)A RNA-binding mode utilized by other YTH domains. Furthermore, the m(6)A pocket cannot bind m(6)A RNA. Our structural and biochemical experiments uncover the mechanism of the YTH domain in binding the DSR motif and help to elucidate the function of Mmi1., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Why do some amyloids cause serious neurodegenerative diseases, while others have important biological functions? A new study of the functional amyloid Orb2 suggests that it's all about speed. Read the Research Article.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

Oocyte developmental competence is the ability of the mature oocyte to be fertilized and subsequently drive early embryo development. Developmental competence is acquired by completion of oocyte maturation, a process that includes nuclear (meiotic) and cytoplasmic (molecular) changes. Given that maturing oocytes are transcriptionally quiescent (as are early embryos), they depend on post-transcriptional regulation of stored transcripts for protein synthesis, which is largely mediated by translational repression and deadenylation of transcripts within the cytoplasm, followed by recruitment of specific transcripts in a spatiotemporal manner for translation during oocyte maturation and early development. Motifs within the 3' untranslated region (UTR) of messenger RNA (mRNA) are thought to mediate repression and downstream activation by their association with binding partners that form dynamic protein complexes that elicit differing effects on translation depending on cell stage and interacting proteins. The cytoplasmic polyadenylation (CP) element, Pumilio binding element, and hexanucleotide polyadenylation signal are among the best understood motifs involved in CP, and translational regulation of stored transcripts as their binding partners have been relatively well-characterized. Knowledge of CP in mammalian oocytes is discussed as well as novel approaches that can be used to enhance our understanding of the functional and contributing features to transcript CP and translational regulation during mammalian oocyte maturation. WIREs RNA 2016, 7:71-89. doi: 10.1002/wrna.1316 For further resources related to this article, please visit the WIREs website., (© 2015 Wiley Periodicals, Inc.)

In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these "decay-promoting" introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Memories are thought to be formed in response to transient experiences, in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, we find that the monomeric form of Orb2 represses translation and removes mRNA poly(A) tails, while the oligomeric form enhances translation and elongates the poly(A) tails and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative poly(A) binding protein CG4612 promotes oligomeric Orb2-dependent translation. Our data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA's interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1's depth of nuclear invasion, potentially aiding the virus's integration into gene-dense regions., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Most mammalian genes produce transcripts whose 3' ends are processed at multiple alternative positions by cleavage/polyadenylation (CPA). Poly(A) site cleavage frequently occurs cotranscriptionally and is facilitated by CPA factor binding to the RNA polymerase II (Pol II) C-terminal domain (CTD) phosphorylated on Ser2 residues of its heptad repeats (YS2PTSPS). The function of cotranscriptional events in the selection of alternative poly(A) sites is poorly understood. We investigated Pol II pausing, CTD Ser2 phosphorylation, and processing factor CstF recruitment at wild-type and mutant IgM transgenes that use alternative poly(A) sites to produce mRNAs encoding the secreted and membrane-bound forms of the immunoglobulin (Ig) heavy chain. The results show that the sites of Pol II pausing and processing factor recruitment change depending on which poly(A) site is utilized. In contrast, the extent of Pol II CTD Ser2 phosphorylation does not closely correlate with poly(A) site selection. We conclude that changes in properties of the transcription elongation complex closely correlate with utilization of different poly(A) sites, suggesting that cotranscriptional events may influence the decision between alternative modes of pre-mRNA 3' end processing., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Glioma stem cells derived from primary cultures were divided into an experiment group, a control group, and a blank group and subjected to cytoplasmic polyadenilation element-binding protein (CPEBs) interference, transfection with empty vector, and normal culture, respectively, to compare their invasion abilities. Western blotting showed that siRNA-3 had the strongest interfering effect on CPEBs. CPEBs were expressed in the experiment group with green fluorescence at an expression rate of over 70%. Significantly lower CPEB expression was observed in the experiment group compared to in the control and blank groups (P < 0.05). After 48-h treatment, the apoptotic rate in the experiment group was 21.43%, which was significantly higher than that in the blank (0.51%) and control (1.43%) groups (P < 0.05). After 3 days of treatment, the experiment group grew significantly more slowly than did the control and blank groups (P < 0.05). The transwell invasion assay showed that significantly fewer cells in the experiment group penetrated the membrane than did cells in the control and blank groups (P < 0.05). After CPEB interference, the growth, proliferation, and invasion of glioma stem cells were substantially inhibited, providing support for targeted therapy of glioma and for improving prognosis.

The Cytoplasmic Polyadenylation Element Binding proteins are RNA binding proteins involved in the translational regulation of mRNA. During cell cycle progression, CPEB1 is labeled for degradation by phosphorylation-dependent ubiquitination by the SCF(β-TrCP) ligase. The peptidyl-prolyl isomerase Pin1 plays a key role in CPEB1 degradation. Conditioned by the cell cycle stage, CPEB1 and Pin1 interactions occur in a phosphorylation-independent or -dependent manner. CPEB1 contains six potential phosphorylatable Pin1 binding sites. Using a set of biophysical techniques, we discovered that the pS210 site is unique, since it displays binding activity not only to the WW domain but also to the prolyl-isomerase domain of Pin1. The NMR structure of the Pin1 WW-CPEB1 pS210 (PDB ID: 2n1o) reveals that the pSerPro motif is bound in trans configuration through contacts with amino acids located in the first turn of the WW domain and the conserved tryptophan in the β3-strand. NMR relaxation analyses of Pin1 suggest that inter-domain flexibility is conferred by the modulation of the interaction with peptides containing the pS210 site, which is essential for degradation.

Unlabelled: During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution., Importance: The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3' end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272-1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3' end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3' end processing similar to RNAi-depletion of histone-specific 3' end processing factors FLASH, SLBP, and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3' end processing of histone mRNAs., (© 2015 Michalski and Steiniger; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Canonical histones are synthesized with a peak in S-phase, whereas histone variants are formed throughout the cell cycle. Unlike messenger RNA (mRNA) for all other genes with a poly(A) tail, canonical histone mRNAs contain a stem-loop structure at their 3'-ends. This stem-loop structure is the binding site for the stem-loop binding protein (SLBP), a protein involved in canonical histone mRNA processing. Recently, we found that arsenic depletes SLBP by enhancing its proteasomal degradation and epigenetically silencing the promoter of the SLBP gene. The loss of SLBP disrupts histone mRNA processing and induces aberrant polyadenylation of canonical histone H3.1 mRNA. Here, we present new data supporting the idea that the lack of SLBP allows the H3.1 mRNA to be polyadenylated using the downstream poly(A) signal. SLBP was also depleted in arsenic-transformed bronchial epithelial cells (BEAS-2B), which led us to hypothesize the involvement of SLBP and polyadenylated H3.1 mRNA in carcinogenesis. Here, for the first time, we report that overexpression of H3.1 polyadenylated mRNA, and knockdown of SLBP enhances anchorage-independent cell growth. A pcDNA-H3.1 vector with a poly(A) signal sequence was stably transfected into BEAS-2B cells. Polyadenylated H3.1 mRNA and exogenous H3.1 protein levels were significantly increased in cells containing the pcDNA-H3.1 vector. A soft agar assay revealed that cells containing the vector formed significantly higher numbers of colonies compared to wild-type cells. Moreover, small hairpin RNA for SLBP (shSLBP) was used to knockdown the expression of SLBP. Cells stably transfected with the shSLBP vector grew significantly more colonies in soft agar than cells transfected with a control vector. These data suggest that upregulation of polyadenylated H3.1 mRNA holds potential as a mechanism to facilitate carcinogenesis by toxicants such as arsenic that depletes SLBP.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with β-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1., (© 2014 Wiley Periodicals, Inc.)

In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo., (© 2015 Grzechnik et al.; Published by Cold Spring Harbor Laboratory Press.)

HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.

Platelet derived growth factor-α (PDGFR-α) is expressed in peripheral T cell lymphoma, not otherwise specified (PTCL, NOS). Imatinib mesylate demonstrated in vitro cytotoxicity against primary PTCL, NOS cells. We initiated a trial of imatinib in 12 patients with relapsed or refractory T-cell non-Hodgkin lymphoma (T-NHL). PDGFR-α expression by immunohistochemistry and fluorescence in situ hybridization (FISH) to assess for FIP1L1-PDGFR-α fusion and/or PDGFR-α amplification were not required for study entry. We documented no objective responses. The median progression-free survival was 21.0 days (90% confidence interval [CI] 15.0, 28.0) and median overall survival was 154 days (90% CI 35, 242). Four patients had tissue available for analysis of PDGFR-α by immunohistochemistry and three of these patients' tumors expressed PDGFR-α. Imatinib was not effective for the treatment of peripheral T cell lymphoma in an unselected group of patients in which PDGFR-α expression was not required for study entry.

Defining the RNA target selectivity of the proteins regulating mRNA metabolism is a key issue in RNA biology. Here we present a novel use of principal component analysis (PCA) to extract the RNA sequence preference of RNA binding proteins. We show that PCA can be used to compare the changes in the nuclear magnetic resonance (NMR) spectrum of a protein upon binding a set of quasi-degenerate RNAs and define the nucleobase specificity. We couple this application of PCA to an automated NMR spectra recording and processing protocol and obtain an unbiased and high-throughput NMR method for the analysis of nucleobase preference in protein-RNA interactions. We test the method on the RNA binding domains of three important regulators of RNA metabolism., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribonucleoprotein complexes involved in pre-mRNA splicing and mRNA decay are often regulated by phosphorylation of RNA-binding proteins. Cells use phosphorylation-dependent signaling pathways to turn on and off gene expression. Not much is known about how phosphorylation-dependent signals transmitted by exogenous factors or cell cycle checkpoints regulate RNA-mediated gene expression at the atomic level. Several human diseases are linked to an altered phosphorylation state of an RNA binding protein. Understanding the structural response to the phosphorylation "signal" and its effect on ribonucleoprotein assembly provides mechanistic understanding, as well as new information for the design of novel drugs. In this review, I highlight recent structural studies that reveal the mechanisms by which phosphorylation can regulate protein-protein and protein-RNA interactions in ribonucleoprotein complexes.

Although recent studies have shown that adenosine-to-inosine (A-to-I) RNA editing occurs in microRNAs (miRNAs), its effects on tumour growth and metastasis are not well understood. We present evidence of CREB-mediated low expression of ADAR1 in metastatic melanoma cell lines and tumour specimens. Re-expression of ADAR1 resulted in the suppression of melanoma growth and metastasis in vivo. Consequently, we identified three miRNAs undergoing A-to-I editing in the weakly metastatic melanoma but not in strongly metastatic cell lines. One of these miRNAs, miR-455-5p, has two A-to-I RNA-editing sites. The biological function of edited miR-455-5p is different from that of the unedited form, as it recognizes a different set of genes. Indeed, wild-type miR-455-5p promotes melanoma metastasis through inhibition of the tumour suppressor gene CPEB1. Moreover, wild-type miR-455 enhances melanoma growth and metastasis in vivo, whereas the edited form inhibits these features. These results demonstrate a previously unrecognized role for RNA editing in melanoma progression.

Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. We show that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lig, another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. We further show that Lig forms a complex with Orb2. We infer that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.

Obtaining a precise characterization of eosinophilia is crucial, as successful treatment relies on the underlying etiology of the disease. Platelet-derived growth factor receptor alpha-related disorders were first specified in 2008 as a distinct group of clonal eosinophilic disorders with exceptional responsiveness to imatinib. We herein present the case of a man with myeloid neoplasm and eosinophilia in whom a definitive diagnosis could not be adequately made based on histopathological features who was ultimately diagnosed only after extensive molecular analyses and successfully treated with imatinib. In addition, we discuss the diagnostic and therapeutic approaches to treating patients presenting with eosinophilia.