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Background Long noncoding RNAs (lncRNAs) have driven research focused on their effects as oncogenes or tumor suppressors involved in carcinogenesis. However, the functions and mechanisms of most lncRNAs in colorectal cancer (CRC) remain unclear. Methods The expression of DLGAP1-AS2 was assessed by quantitative RT-PCR in multiple CRC cohorts. The impacts of DLGAP1-AS2 on CRC growth and metastasis were evaluated by a series of in vitro and in vivo assays. Furthermore, the underlying mechanism of DLGAP1-AS2 in CRC was revealed by RNA pull down, RNA immunoprecipitation, RNA sequencing, luciferase assays, chromatin immunoprecipitation, and rescue experiments. Results We discovered that DLGAP1-AS2 promoted CRC tumorigenesis and metastasis by physically interacting with Elongin A (ELOA) and inhibiting its protein stability by promoting tripartite motif containing 21 (Trim21)-mediated ubiquitination modification and degradation of ELOA. In particular, we revealed that DLGAP1-AS2 decreases phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) expression by inhibiting ELOA-mediated transcriptional activating of LHPP and thus blocking LHPP-dependent suppression of the AKT signaling pathway. In addition, we also demonstrated that DLGAP1-AS2 was bound and stabilized by cleavage and polyadenylation specificity factor (CPSF2) and cleavage stimulation factor (CSTF3). Conclusions The discovery of DLGAP1-AS2, a promising prognostic biomarker, reveals a new dimension into the molecular pathogenesis of CRC and provides a prospective treatment target for this disease.

Regulated gene expression is largely achieved by controlling the activities of essential, multisubunit RNA polymerase transcription elongation complexes (TECs). The extreme stability required of TECs to processively transcribe large genomic regions necessitates robust mechanisms to terminate transcription. Efficient transcription termination is particularly critical for gene-dense bacterial and archaeal genomes1-3 in which continued transcription would necessarily transcribe immediately adjacent genes and result in conflicts between the transcription and replication apparatuses4-6; the coupling of transcription and translation7,8 would permit the loading of ribosomes onto aberrant transcripts. Only select sequences or transcription termination factors can disrupt the otherwise extremely stable TEC and we demonstrate that one of the last universally conserved archaeal proteins with unknown biological function is the Factor that terminates transcription in Archaea (FttA). FttA resolves the dichotomy of a prokaryotic gene structure (operons and polarity) and eukaryotic molecular homology (general transcription apparatus) that is observed in Archaea. This missing link between prokaryotic and eukaryotic transcription regulation provides the most parsimonious link to the evolution of the processing activities involved in RNA 3'-end formation in Eukarya.

RNA-binding proteins (RBPs) are key regulators of gene expression. RBP dysregulation is reported to play essential roles in tumorigenesis. However, the role of RBPs in urothelial carcinoma of the bladder (UCB) is only starting to be unveiled. Here, we comprehensively assessed the mRNA expression landscape of 104 RBPs from two independent UCB cohorts, Sun Yat-sen University Cancer Center (SYSUCC) and The Cancer Genome Atlas (TCGA). Fragile X-related gene 1 (FXR1) was identified as a novel cancer driver gene in UCB. FXR1 overexpression was found to be related to the poor survival rate in the SYSUCC and TCGA cohorts. Functionally, FXR1 promotes UCB proliferation and tumorigenesis. Mechanistically, FXR1 serves as a platform to recruit CFIm25 and CFIm68, forming a novel 3′ processing machinery that functions in sequence-specific poly(A) site recognition. FXR1 affects the 3′ processing of Tumor necrosis factor receptor-associated factor 1 (TRAF1) mRNA, which leads to nuclear stabilization. The novel regulatory relationship between FXR1 and TRAF1 can enhance cell proliferation and suppress apoptosis. Our data collectively highlight the novel regulatory role of FXR1 in TRAF1 3′ processing as an important determinant of UCB oncogenesis. Our study provides new insight into RBP function and provides a potential therapeutic target for UCB.

Predicting the pathogenicity of biallelic missense variants can be challenging. Here, we use a deficit of observed homozygous carriers of missense variants, versus an expected number in a set of 153,054 chip-genotyped Icelanders, to identify potentially pathogenic genotypes. We follow three missense variants with a complete deficit of homozygosity and find that their pathogenic effect in homozygous state ranges from severe childhood disease to early embryonic lethality. One of these variants is in CPSF3, a gene not previously linked to disease. From a set of clinically sequenced Icelanders, and by sequencing archival samples targeted through the Icelandic genealogy, we find four homozygous carriers. Additionally, we find two homozygous carriers of Mexican descent of another missense variant in CPSF3. All six homozygous carriers of missense variants in CPSF3 show severe intellectual disability, seizures, microcephaly, and abnormal muscle tone. Here, we show how the absence of certain homozygous genotypes from a large population set can elucidate causes of previously unexplained recessive diseases and early miscarriage.

Alternative polyadenylation (APA) has emerged as a prevalent feature associated with cancer development and progression. The advantage of APA to tumor progression is to induce oncogenes through 3'-UTR shortening, and to inactivate tumor suppressor genes via the re-routing of microRNA competition. We previously identified the Mammalian Cleavage Factor I-25 (CFIm25) (encoded by Nudt21 gene) as a master APA regulator whose expression levels directly impact the tumorigenicity of glioblastoma (GBM) in vitro and in vivo. Despite its importance, the role of Nudt21 in GBM development is not known and the genes subject to Nudt21 APA regulation that contribute to GBM progression have not been identified. Here, we find that Nudt21 is reduced in low grade glioma (LGG) and all four subtypes of high grade glioma (GBM). Reduced expression of Nudt21 associates with worse survival in TCGA LGG cohorts and two TCGA GBM cohorts. Moreover, although CFIm25 was initially identified as biochemically associated with both CFIm59 and CFIm68, we observed three CFIm distinct subcomplexes exist and CFIm59 protein level is dependent on Nudt21 expression in GBM cells, but CFIm68 is not, and that only CFIm59 predicts prognosis of GBM patients similar to Nudt21. Through the use of Poly(A)-Click-Seq to characterize APA, we define the mRNAs subject to 3'-UTR shortening upon Nudt21 depletion in GBM cells and observed enrichment in genes important in the Ras signaling pathway, including Pak1. Remarkably, we find that Pak1 expression is regulated by Nudt21 through its 3'-UTR APA, and the combination of Pak1 and Nudt21 expression generates an even stronger prognostic indicator of GBM survival versus either value used alone. Collectively, our data uncover Nudt21 and its downstream target Pak1 as a potential "combination biomarker" for predicting prognosis of GBM patients.

Long non-coding RNAs (lncRNAs) play critical roles in regulation of immunological pathways. Consequently, their expression profile represents new biomarkers for susceptibility and progression of immunological disorders. However, their role in chronic inflammatory diseases such as multiple sclerosis (MS) remained unknown. Here, we assessed the expression of lncRNAs MALAT1 and HOTAIRM1 as well as their target genes in peripheral blood of MS patients to show their possible roles in disease initiation and progression. In this study, 50 patients with relapsing-remitting MS and 50 healthy matched controls were enrolled. Comparative Ct method via TaqMan assay was used to quantify transcript levels of MALAT1, HOTAIRM1, AGO2, CSTF2, CPSF7, and WDR33. Our analysis depicted significant differences in lncRNAs and their target genes expression levels. AGO2 expression was significantly elevated in MS patients (P

Background The human myxovirus resistance 2 (Mx2/MxB) protein was originally found to regulate cytoplasmic-nuclear transport but was recently reported to restrict HIV-1 replication by binding to HIV-1 capsid (CA), preventing uncoating, the nuclear import of pre-integration complex (PIC) and viral DNA integration. This work explores the mechanisms of MxB-mediated HIV-1 inhibition. Results We demonstrated that MxB represses NUP358-mediated PIC nuclear import and HIV-1 replication. Moreover, MxB’s effects on PIC nuclear import and HIV-1 replication depend critically on cofactor cleavage and polyadenylation specificity factor subunit 6 (CPSF6). MxB binds nucleoporin NUP358, blocks NUP358-CA interaction, thereby impeding the nuclear import of HIV-1 PIC with CPSF6 binding to PIC. More intriguingly, CPSF6’s role in nuclear import depends on MxB, being a facilitator of HIV-1 nuclear import on its own, but becoming an inhibitor when MxB is present. Conclusions Our work establishes that MxB impedes the NUP358-mediated HIV-1 nuclear import and viral replication cooperatively with CPSF6.

Background Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer-related death worldwide. Increasing evidence indicates that the deregulation of long noncoding RNAs (lncRNAs) contributes to tumor initiation and progression; however, little is known about the biological role of cancer susceptibility candidate 9 (CASC9) in CRC. Methods Novel lncRNAs potentially involved in CRC tumorigenesis were identified from datasets downloaded from The Cancer LncRNome Atlas and The Atlas of Noncoding RNAs in Cancer. The CRC cell lines HCT-116, HCT-116 p53−/−, SW620, SW480, HT-29, LoVo, LS-174T, and RKO were used. Colony-formation, MTS, cell-cycle, apoptosis, and in-vivo tumorigenesis assays were used to determine the role of CASC9 in CRC cell growth in vitro and in vivo. Potential interaction between CASC9 and cleavage and polyadenylation specificity factor subunit 3 (CPSF3) was evaluated using RNA immunoprecipitation and RNA-protein pull-down assays. RNA-sequencing was performed to analyze gene expression following CASC9 knockdown. RT-qPCR, western blotting, and mRNA decay assays were performed to study the mechanisms involved. Results CASC9 was frequently upregulated in CRC, which was correlated with advanced TNM stage, and higher CASC9 levels were associated with poor patient outcomes. Knockdown of CASC9 inhibited growth and promoted apoptosis in CRC cells, whereas ectopic CASC9 expression promoted cell growth in vitro and in vivo. We demonstrated that CPSF3 is a CASC9-interacting protein, and knockdown of CPSF3 mimicked the effects of CASC9 knockdown in CRC cells. Furthermore, we found that CASC9 exerts its oncogenic activity by modulating TGFβ2 mRNA stability and upregulating the levels of TGFβ2 and TERT, resulting in an increase in phosphorylated SMAD3 and activation of TGF-β signaling, and enhanced TERT complex function in CRC cells. Finally, CPSF3 was significantly upregulated in CRC tissues as compared with adjacent or non-adjacent normal colon tissues, and CASC9, CPSF3, and TGFβ2 levels in human CRC tissues were positively correlated. Conclusions CASC9 is a promising prognostic predictor for patients with CRC and the CASC9-CPSF3-TGFβ2 axis is a potential therapeutic target for CRC treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1263-3) contains supplementary material, which is available to authorized users.

Trichomonas vaginalis is the causative agent of trichomonosis, a sexually transmitted disease (STD) that affects over 180 million people worldwide. This parasite is capable to infect the urogenital tract of women and men, both microenvironments might affect the expression of key genes that may be involved in the parasite pathogenesis. The processing of 3′ end of mRNA promotes mRNA stability in many eukaryotes, however in T. vaginalis this molecular machinery is under research. By means of an in silico analysis we identified putative proteins of the 3′ end mRNA processing machinery of T. vaginalis, and by RT-PCR assays we evaluated the expression of eight of these genes in a female and male T. vaginalis isolates. According to the in silico analysis, the T. vaginalis 3′ end mRNA processing machinery, comprises a similar complex and protein factors that those described in Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae and Entamoeba histolytica. The complex contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CFIm) and cleavage factor II (CFIIm). We demonstrated that genes tvpsf2p, tvcfi25, tvcpsf160, tvcpsf73, tvfip1, tvpap1, tvpc4 and tvpabp are expressed in male or female T. vaginalis isolates. Besides we identify two different isoforms of TvPC4. T. vaginalis genome contains most of genes encoding for 3′ end mRNA processing, which may be transcriptionally active and could be involved in the capping, splicing, cleavage and polyadenylation of mRNAs in this parasite. Further studies are necessary to elucidate the biological meaning of our findings.

Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes–substrates or enzymes–nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA–protein interactions, we used 2′-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of V max and K m have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'-OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.

Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3′-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF Im), and cleavage factor II (CF IIm). Additional protein factors include poly(A) polymerase (PAP), poly(A) binding protein (PABP), symplekin, and the C-terminal domain (CTD) of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA (CF IA), cleavage factor IB (CF IB), and cleavage and polyadenylation factor (CPF).

Eukaryotic poly(A) signals direct mRNA 3'-end processing and also pausing and termination of transcription. We show that pausing and termination require the processing factor CPSF, which binds the AAUAAA hexamer of the mammalian poly(A) signal. Pausing does not require the RNA polymerase II C-terminal domain (CTD) or the cleavage stimulation factor, CstF, that binds the CTD. Pull-down experiments show that CPSF binds, principally through its 30-kDa subunit, to the body of the polymerase. CPSF can also bind CstF, but this seems to be mutually exclusive with polymerase binding. We suggest that CPSF, while binding the body of the polymerase, scans for hexamers in the extruding RNA. Any encounter with a hexamer triggers pausing. If the hexamer is part of a functional poly(A) signal, CstF is recruited and binds CPSF, causing it to release the polymerase body and move (with CstF) to the CTD.

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is upregulated in multiple malignancies and is associated with poor prognosis. While thepro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associatedcarboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavagegenerating MUC16-Cter has remained unknown. Using different lengths of dual-epitope(N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, wedemonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretchof twelve amino acids that generates a ~17 kDa cleaved product and isdistinct from the predicted sites. This was further corroborated by domain swappingexperiment. Further, the cleavage of MUC16 was found to take place in theGolgi/post-Golgi compartments and is dependent on the acidic pH in the secretorypathway. A similar pattern of ~17 kDa cleaved MUC16 was observed inmultiple cell types eliminating the possibility of cell type specific phenomenon.MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds tothe chromatin suggesting its involvement in regulation of gene expression. Takentogether, we demonstrate for the first time the oft-predicted cleavage of MUC16 thatis critical in designing successful therapeutic interventions based on MUC16.

Heat-stable protein fraction in seeds is believed to enrich many proteins functioning in the acquisition of stress-tolerance of seeds. In this study, the composition of heat-stable protein fraction in imbibed cowpea (Vigna unguiculata) seeds was analyzed by SDS-PAGE and two-dimensional gel electrophoresis coupled with mass spectrometry. The results indicated that approximately 12.4 % of seed soluble proteins were stable after heat treatment at 100 °C for 10 min. Twenty-two putative heat-stable proteins were identified using MALDI-TOF/TOF MS. Most of these heat-stable proteins were late embryogenesis abundant proteins, and there were other stress-related proteins including Cu/Zn superoxide dismutase and 17.4 kDa Class I heat-shock protein. A cyclophilin protein, a cleavage and polyadenylation specificity factor and a Pumilio-family RNA binding protein were also present in the heat-stable fraction. The identified heat-stable proteins were more hydrophilic proteins and may accumulate to stabilize cellular components and maintain seed viability during seed development and germination.

The formation of eukaryotic mRNAs involves the cleavage and polyadenylation of pre- mRNAs. To investigate the sequence requirement of putative polyadenylation signals (PASs), poly(A) sites and downstream elements (DUEs) in 3′-end-pro- cessing in rice, we compared expressed sequences tags (ESTs) with poly(A) extremity to full-length cDNA sequences and constructed a database of 12969 pre- mRNA sequences in ?40―+40 nt surrounding the poly(A) sites, which were from 9953 genes. The al- ternative poly(A) sites were revealed in approxi- mately 25% of mRNAs. Nearly 80% of pre-mRNAs showed stringent requirement of the YA (CA or UA) at poly (A) sites for polyadenylation. About 7.9% had the AAUAAA signals on ?40―?1 nt upstream of the poly(A) sites. Over 60% of mRNAs probably used the one- or two-base variants of AAUAAA hexamers as their PASs in 3′ fragments. The single-base variants of AAUGAA revealed the high frequency in 11.5% of 3′ fragments. The DUEs were detected in 90% of pre- mRNAs, especially more than half of the pre-mRNAs with multi-base variants of AAUAAA had the DUEs surrounding the poly(A) site. The location of DUE is also important for defining the cleavage site. Al- though most of the rice pre-mRNAs did not contain AAUAAA signal, the existence of downstream ele- ments ensured the efficiency of cleavage-polyade- nylation

Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry-compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation.

The Arabidopsis genome possesses a number of sequences that are predicted to encode proteins that are similar to mammalian and yeast polyadenylation factor subunits. One of these resides on chromosome V and has the potential to encode a polypeptide related to the 100 kDa subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF). This gene encodes a ca. 2400 nucleotide mRNA that in turn can be translated to yield a polypeptide that is 39% identical to the mammalian CPSF100 protein. Antibodies raised against the Arabidopsis protein recognized distinctive polypeptides in nuclear extracts prepared from pea and wheat germ, consistent with the hypothesis that the Arabidopsis protein is resident in a nuclear polyadenylation complex. Interestingly, the Arabidopsis CPSF100 was found to interact with a portion of a nuclear poly(A) polymerase. This interaction was attributable to a 60 amino acid domain in the CPSF100 polypeptide and the N-terminal 220 amino acids of the poly(A) polymerase. An analogous interaction has yet to be described in other eukaryotes. The interaction with PAP thus indicates that the plant CPSF100 polypeptide is likely part of the 3'-end processing machinery, but suggests that this complex may function differently in plants than it does in mammals and yeast.

Transcription termination by RNA polymerase II (Pol II) on most mRNA-encoding genes is dependent on transcription through a functional poly(A) signal. One model to explain this dependence predicts co-trancriptional cleavage of RNA at the poly(A) site. Electron microscopic (EM) visualization was used to investigate the in vivo frequency of transcript cleavage prior to termination. Over 100 unidentified Drosophila Pol II-transcribed genes were analyzed. Although some genes exhibited cleaved transcripts near their 3' ends, and some had a lower polymerase density at their 3' end relative to the rest of the gene, the majority of genes (64%) had uncleaved transcripts and no change in polymerase density at the 3' end, consistent with release of full-length transcripts at a discrete site. Thus, in Drosophila, cleavage at the poly(A) site sometimes occurs co-transcriptionally, but does not appear to be a prerequisite to termination. Next, two components of the polyadenylation complex were immunolocalized on polytene chromosomes and were found to differ in distribution both qualitatively and quantitatively. The EM results indicate that co-transcriptional recognition of the poly(A) signal, which is required for termination, does not equate with co-transcriptional cleavage, and the immunofluorescence results suggest that this may be due to incomplete or nonstoichiometric assembly of the polyadenylation machinery on nascent transcripts.

Proteolytic processing of the mouse hepatitis virus strain A59 (MHV-A59) replicase gene product, pp1a, results in polypeptides p28, p65, p50, and p240 in infected cells. Based on previously identified p28 and p65 cleavage sites, a p50 cleavage site was proposed to occur between Ala-1262 and Ala-1263. Results of mutagenesis and in vitro cleavage assays show that PLP-1 was able to cleave in trans when the proposed p50 cleavage sequence replaced the p28 cleavage sequence. Mutagenesis was also used to investigate cleavage between Gly-904 and Val-905, a cleavage site predicted to produce a precursor of p65, p72, that was detected in cells infected with MHV strain JHM, but not with MHV-A59. No cleavage could be detected using substrate that carried both the p65 site and the predicted p72 cleavage sequence. Thus, it appeared that PLP-1 could recognize the proposed p50 sequence but not the predicted p72 site under the in vitro conditions used.

Background The influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES). Methods In order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence. Results NS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Impaired activation of Akt phosphorylation by the replication-deficient viruses indicates possible disruption of NS1-p85β interaction by mutations in the NES region. Conclusion We conclude that mutations within the NS1 NES result in impairment of several NS1 functions which extends further from the NES site being only involved in regulating the nuclear-cytoplasmic trafficking of NS1.

Inhibition of CPSF30 function by the effector domain of influenza A virus of non-structural protein 1 (NS1A) protein plays a critical role in the suppression of host key antiviral response. The CPSF30-binding site of NS1A appears to be a very attractive target for the development of new drugs against influenza A virus. In this study, structure-based molecular docking was utilized to screen more than 30,000 compounds from a Traditional Chinese Medicine (TCM) database. Four drug-like compounds were selected as potential inhibitors for the CPSF30-binding site of NS1A. Docking conformation analysis results showed that these potential inhibitors could bind to the CPSF30-binding site with strong hydrophobic interactions and weak hydrogen bonds. Molecular dynamics simulations and MM-PBSA calculations suggested that two of the inhibitors, compounds 32056 and 31674, could stably bind to the CPSF30-binding site with high binding free energy. These two compounds could be modified to achieve higher binding affinity, so that they may be used as potential leads in the development of new anti-influenza drugs.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed. Cell Death and Differentiation (2000) 7, 628–636

Here we identify the hematopoietic proto-oncogene Vav1 as a caspase substrate during apoptosis in lymphoid cells. Cleavage of Vav1 is prevented by the caspase inhibitors zDEVD and zVAD as well as by expression of CrmA. Vav1 is cleaved in vivo at the evolutionary conserved caspase consensus cleavage site DLYD161C, generating the carboxy-terminal cleavage product Vav1p76 of intermediate stability. In vitro caspase assays reveal cleavage of Vav1 at position 161 either by apoptotic cell lysates or by recombinant caspase-3. Mutation of Asp 161 to Ala leads to the usage of the adjacent alternative cleavage sequence DQID150D. Mutation of both cleavage sites at position 150 and 161 protects Vav1 from caspase-mediated proteolysis in vitro and in vivo. The cleavage product Vav1p76 is capable of activating JNK in T-cells, but fails to induce the phosphorylation of p38/HOG1. Vav1p76 displays a diminished capacity to activate the transcription factors NF-AT, AP-1 and NF-kappaB, and thus completely fails to activate IL-2 transcription. Since Vav1 is essential for IL-2 production and plays a central role for cytoskeletal reorganization, its proteolytic inactivation during apoptosis affects multiple downstream targets.

Production of messenger RNA in eukaryotic cells is a complex, multistep process. mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase. These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation. Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo. For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation. These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation. Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.

Background The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. Methods NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. Results Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively. Conclusions The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

5’-capping, splicing and 3’-cleavage/polyadenylation of a nascent RNA transcript are all coupled to transcription. Transcription is regulated by the post-translational modification of histones at the chromatin template. How histone modifications affect cotranscriptional RNA processing, however, is not well understood. By studying how the chromatin template contributes to the dynamic gene expression induced downstream of the JAK-STAT signaling pathway, we have uncovered relationships between specific histone modifications and RNA processing. Here we report that H2B monoubiquitination (ubH2B) is required for the effective 3’-end processing of the JAK-STAT inducible transcript, IRF1. RNAi-mediated depletion of the ubiquitin hydrolase, USP22 increases ubH2B levels and decreases transcriptional elongation at IRF1. Unexpectedly, 3’-end cleavage and polyadenylation of IRF1 is diminished, leading to a 2-to-3-fold increase in unprocessed IRF1 transcripts. The polyadenylation factor, CPSF73 is not effectively recruited and serine 2 phosphorylation (Ser2P) of the C-terminal domain of RNA polymerase II is disrupted. Two other JAK-STAT inducible transcripts are similarly affected, while two constitutively expressed transcripts are not. A working model, wherein a cycle of H2B ubiquitination/deubiquitination specifies Ser2P to regulate elongation and 3’-end processing of JAK-STAT inducible mRNAs is proposed. These results further elaborate USP22 function and its role as a putative cancer stem cell marker.

Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5' end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5'-(gamma-thio)triphosphate (ATP gamma S), replacement of RecA protein by a mutant (RecA1) protein, or an increase in Mg2+ concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.

Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Background Alternative polyadenylation is one of the mechanisms in human cells that give rise to a variety of transcripts from a single gene. More than half of the human genes have multiple polyadenylation sites (poly(A) sites), leading to variable mRNA and protein products. Previous studies of individual genes have indicated that alternative polyadenylation could occur in a tissue-specific manner.

We have introduced chimeric genes containing polyadenylation signals from a human gene and two animal virus genes into tobacco cells. We see, in all three cases, inefficient and 'aberrant' utilization of the foreign polyadenylation signals. We find that a chimeric gene carrying the polyadenylation site of the human growth hormone gene is polyadenylated at three sites in the vicinity of the site that is polyadenylated in human cells. A chimeric gene containing the polyadenylation site from the adenovirus 5 E1A gene is polyadenylated at a site 11 bases downstream from that reported in animal cells. A gene carrying the polyadenylation site from the SV40 early region is polyadenylated some 80 bases upstream from the site that is polyadenylated in animal cells. In all three cases, related mRNAs ending at flanking 'authentic' plant polyadenylation sites can be detected, indicating that the foreign polyadenylation signals are inefficiently utilized in tobacco cells.

Mammalian cells selected to resist N-(phosphonacetyl)-L-aspartate (PALA) contain amplified copies of the CAD gene. While a single 7.9-kb mRNA species is detected in PALA-sensitive and most PALA-resistant cell lines, two RNA species (7.9 and 10.2 kb) are detected in two related drug-resistant mutants presumably derived from the same parental cell. In this report we show that the 10.2-kb RNA is produced as a direct consequence of a sequence rearrangement adjacent to the 3′ end of the CAD gene in these cell lines. A CAD gene containing the sequence rearrangement was cloned from one of these lines and found to produce both RNA species when transfected into CAD-deficient cells. DNA sequencing and S1 analysis demonstrate that the 10.2-kb RNA is produced by alternative polyadenylation rather than by alternative splicing. Sequence analysis also reveals that several consensus poly(A) addition signals (AATAAA) were brought into close proximity to the CAD gene by virtue of the rearrangement. While sequences adjacent to each of the polyadenylation signals contain additional features postulated to be important for the selection of the site of poly (A) addition, S1 mapping analysis indicates that only one of the polyadenylation signals is used. A comparison of all of these sites suggests that multiple sequence motifs are required to form a functional polyadenylation and cleavage signal.