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Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

Background Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. Methods Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Results The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. Conclusions Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors., Suppression of terminal differentiation is essential for epidermal progenitor maintenance. Here, the authors show that intronic polyadenylation is dynamically regulated by the cooperation between CPSF and RNA-binding proteins to influence epidermal differentiation gene expression.

Significance For several decades, retroviral core uncoating has been thought to occur in the cytoplasm in coordination with reverse transcription, and while some recent studies have concluded that HIV-1 uncoating occurs at the nuclear envelope during nuclear import, none have concluded that uncoating occurs in the nucleus. Here, we developed methods to study HIV-1 uncoating by direct labeling and quantification of the viral capsid protein associated with infectious viral cores that produced transcriptionally active proviruses. We find that infectious viral cores in the nuclei of infected cells are largely intact and uncoat near their integration sites just before integration. These unexpected findings fundamentally change our understanding of HIV-1 postentry replication events., HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3′end-sequencing data of Methanococcus maripaludis, found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3′-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

Host shutoff in influenza A virus (IAV) infection is a key process contributing to viral takeover of the cellular machinery and resulting in the downregulation of host gene expression. Analysis of nascently transcribed RNA in a cellular model that allows the functional induction of NS1 demonstrates that NS1 suppresses host transcription. NS1 inhibits the expression of genes driven by RNA polymerase II as well as RNA polymerase I-driven promoters, but not by the noneukaryotic T7 polymerase. Additionally, transcriptional termination is deregulated in cells infected with wild-type IAV. The NS1 effector domain alone is able to mediate both effects, whereas NS1 mutant GLEWN184-188RFKRY (184-188) is not. Overexpression of CPSF30 counteracts NS1-mediated inhibition of RNA polymerase II-driven reporter gene expression, but knockdown of CPSF30 expression does not attenuate gene expression. Although NS1 is associated with nuclear chromatin, superresolution microscopy demonstrates that NS1 does not colocalize with genomic DNA. Moreover, NS1 mutants and NS1 fusion proteins, unable to associate with nuclear chromatin and displaying an altered subcellular distribution are still able to attenuate reporter gene expression. However, tethering NS1 artificially to the cytoskeleton results in the loss of reporter gene inhibition. A NS1 deficient in both native nuclear localization signals (NLS) is able to inhibit gene expression as effective as wild-type NS1 when a synthetic NLS relocates it to specific structures of the nucleus. Colocalization experiments and reporter gene cotransfection experiments with a NS1 fusion guiding it to nuclear speckles suggest that the presence of NS1 in nuclear speckles seems to be essential for host shutoff. IMPORTANCE We investigated the role of IAV nonstructural protein 1 NS1 in host gene shutoff-a central feature of IAV replication. We demonstrate that the effector domain of NS1 alone mediates host gene shutoff by inhibition of host transcription and by deregulation of the polyadenylation (polyA) signal-mediated 3' termination of host transcription. NS1 mutated in amino acids 184 to 188 fails to shut off host gene expression. Knockdown of CPSF30 does not result in transcriptional attenuation. By analyzing the subcellular localization of modified NS1 proteins and relating these data to their ability to inhibit reporter gene expression, we show for the first time that the presence of NS1 in granular structures of the nucleus-representing most likely nuclear speckles-seems to be essential to mediate host gene shutoff. Thus, our data present so far unknown insights into the molecular and spatial requirements needed for IAV-NS1-mediated host shutoff.

HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps., Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages.

Summary: Many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression. : Gaucherand et al. uncover a unique relationship between RNA degradation by the influenza A virus ribonuclease PA-X and host RNA splicing, which allows PA-X to selectively target host RNAs for destruction. Keywords: influenza, host shutoff, PA-X, splicing, CFIm

The HIV-1 latent reservoir is the major barrier to an HIV cure. Due to low levels or lack of transcriptional activity, HIV-1 latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies based on protein expression. To target the latent reservoir, further understanding of HIV-1 proviral transcription is required. In this study, we demonstrate a novel role for cleavage and polyadenylation specificity factor 6 (CPSF6) in HIV-1 transcription. We show that knockout of CPSF6 hinders reactivation of latent HIV-1 proviruses by PMA in primary CD4+ cells. CPSF6 knockout reduced HIV-1 transcription, concomitant with a drastic reduction in the phosphorylation levels of Pol II and CDK9. Knockout of CPSF6 led to abnormal stabilization of protein phosphatase 2A (PP2A) subunit A, which then acted to dephosphorylate CDK9, downmodulating CDK9's ability to phosphorylate the Pol II carboxy-terminal domain. In agreement with this mechanism, incubation with the PP2A inhibitor, LB100, restored HIV-1 transcription in the CPSF6 knockout cells. Destabilization of PP2A subunit A occurs in the ubiquitin proteasome pathway, wherein CPSF6 acts as a substrate adaptor for the ITCH ubiquitin ligase. Our observations reveal a novel role of CPSF6 in HIV-1 transcription, which appears to be independent of its known roles in cleavage and polyadenylation and the targeting of preintegration complexes to the chromatin for viral DNA integration. IMPORTANCE CPSF6 is a cellular factor that regulates cleavage and polyadenylation of mRNAs and participates in HIV-1 infection by facilitating targeting of preintegration complexes to the chromatin. Our observations reveal a second role of CPSF6 in the HIV-1 life cycle that involves regulation of viral transcription through controlling the stability of protein phosphatase 2A, which in turn regulates the phosphorylation/dephosphorylation status of critical residues in CDK9 and Pol II.

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites, however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions.

Cleavage of nascent transcripts is a fundamental process for eukaryotic messenger RNA (mRNA) maturation and for production of different mRNA isoforms. In eukaryotes, cleavage of mRNA precursors by the highly conserved endonuclease CPSF73 is critical for mRNA stability, export from the nucleus, and translation. As an essential enzyme in the cell, CPSF73 surprisingly shows promise as a drug target for specific cancers and for protozoan parasites. In this review, we cover our current understanding of CPSF73 in cleavage and polyadenylation, histone pre-mRNA processing, and transcription termination. We discuss the potential of CPSF73 as a target for novel therapeutics and highlight further research into the regulation of CPSF73 that will be critical to understanding its role in cancer and other diseases.

Background Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. Results Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs. Conclusions We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-021-02298-y.

C.V. was recipient of an FPI-Severo Ochoa Fellowship from the Spanish Ministry of Economy and Competitiveness. Work in J.V. laboratory is supported by the European Research Council (ERC AdvG 670146), AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2017 89308-P) and the Centre of Excellence Severo Ochoa. Work in T.G.'s laboratory was supported by the European Research Council FP7/2007-2013 (ERC Synergy Grant 4D-Genome) the Ministerio de Educación y Ciencia (SAF.2012-37167) and AGAUR. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership and the CERCA Programme / Generalitat de Catalunya. UDTRIAS Background: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.

Alternative polyadenylation (APA) in breast tumor samples results in the removal/addition of cis-regulatory elements such as microRNA (miRNA) target sites in the 3′-untranslated region (3′-UTRs) of genes. Although previous computational APA studies focused on a subset of genes strongly affected by APA (APA genes), we identify miRNAs of which widespread APA events collectively increase or decrease the number of target sites [probabilistic inference of microRNA target site modification through APA (PRIMATA-APA)]. Using PRIMATA-APA on the cancer genome atlas (TCGA) breast cancer data, we found that the global APA events change the number of the target sites of particular microRNAs [target sites modified miRNA (tamoMiRNA)] enriched for cancer development and treatments. We also found that when knockdown (KD) of NUDT21 in HeLa cells induces a different set of widespread 3′-UTR shortening than TCGA breast cancer data, it changes the target sites of the common tamoMiRNAs. Since the NUDT21 KD experiment previously demonstrated the tumorigenic role of APA events in a miRNA dependent fashion, this result suggests that the APA-initiated tumorigenesis is attributable to the miRNA target site changes, not the APA events themselves. Further, we found that the miRNA target site changes identify tumor cell proliferation and immune cell infiltration to the tumor microenvironment better than the miRNA expression levels or the APA events themselves. Altogether, our computational analyses provide a proof-of-concept demonstration that the miRNA target site information indicates the effect of global APA events with a potential as predictive biomarker.

Cleavage factor I m25 is a newly discovered solid tumor-related gene, however, its precise role in cancer pathogenesis has not yet been characterized. Alternative polyadenylation is an RNA-processing mechanism that generates distinct 3′-termini on messenger RNAs, producing messenger RNA isoforms. Different factors influence the initiation and development of this process. As a key factor in alternative polyadenylation, cleavage factor I m25 plays an important role in messenger RNA maturation and cell signal transduction. Moreover, by regulating the process of alternative polyadenylation, it can inhibit the proliferation, invasion, and metastasis of a variety of tumors. Cleavage factor I m25 also acts as an oncogene in select tumors. The present review focuses on the role of cleavage factor I m25 in solid tumors and treatment. Due to the lack of current knowledge regarding the mechanisms of action and regulation of cleavage factor I m25 and alternative polyadenylation, it is necessary to further examine their role in cancer as well as in other diseases.

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci., Early steps of HIV infection of primary human cells remain poorly understood. Here, Francis et al. show that early viral replication complexes accumulate within nuclear speckles, in reliance on viral capsid/host CPSF6 interactions, and preferentially integrate in speckle-associated genomic domains.

Architecture of an mRNA processor The 3′-end processing of the three major classes of RNA polymerase II transcripts in metazoan cells—polyadenylated messenger RNAs (mRNAs), histone mRNAs, and small nuclear RNAs (snRNAs)—requires three distinct machineries that share common features. Sun et al. reconstituted the active human histone pre-mRNA 3′-end processing machinery and solved its structure at near-atomic resolution by cryo–electron microscopy. This structure provides a basis for understanding the mechanism of the shared catalytic reactions between histone pre-mRNA and canonical pre-mRNA and snRNA 3′-end processing machineries. Science , this issue p. 700

Interstitial deletions of 16q chromosome including 16q12.1q21 region are very rare, with only three cases reported to date. Main clinical features include dysmorphisms, short stature, microcephaly, eye abnormalities, epilepsy, development delay, intellectual disability, and autism spectrum disorder. We report two independent subjects with 16q12.1q21 deletion syndrome presenting with dysmorphic facial features, developmental delay, strabismus, and aggressive behavior. A minimal region of overlap spanning 1.7 Mb on chromosome 16, including IRX5, GNAO1, and NUDT21 genes was shared among these two cases and those previously reported. This minimal region of overlap suggests the potential pathogenic role of these genes, previously implicated in diseases of the central nervous system.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.

Background/Aims: PI3KCA and mutant p53 are associated with tumorigenesis and the development of cancers. NVP-BKM120, a selective pan-PI3K inhibitor, exerts the antitumor activity by suppressing the PI3K signaling pathway. Prima-1 Met , a low molecular weight compound, can rescue the gain-of-function of mutant p53 by restoring its transcriptional function. In this study, we investigated whether PI3K inhibition combined with mutant p53 reactivation could enhance the antitumor effect in thyroid cancer cells. Methods: The effects of BKM120 and Prima-1 Met on the proliferation, apoptosis, migration and invasion of thyroid cancer cells were measured by MTT, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Thyroid differentiation was assessed by detecting the expression levels of specific markers using RT-PCR and Western blot. The in vivo antitumor efficacy was analyzed in a mouse xenograft model. Results: The combinational treatment of BKM120 and Prima-1 Met significantly enhanced the inhibitions of cell viability, colony formation, migration and invasion, and the induction of apoptosis in thyroid cell lines, and synergistically suppressed tumor xenograft growth by inhibiting the PI3K/Akt/mTOR and EMT signaling pathways, up-regulating p53 targeted genes, and triggering the release of cytochrome c. Moreover, the combination of BKM120 and Prima-1 Met suppressed the stemlike traits of thyroid cancer cells and promoted their differentiation by upregulating the expression of thyroid-specific differentiation markers and repressing the expression of cancer stem cell markers. Furthermore, the mechanism study demonstrated that the combinational treatment synergistically abrogated the binding of CPSF4 at the promoter of hTERT and thus suppressed hTERT expression. Consistently, overexpression of hTERT rescued the inhibitions of cell viability, invasion and stem-like traits mediated by the combination of BKM120 and Prima-1 Met . Conclusion: Our results showed that the combination of BKM120 with Prima-1 Met synergistically suppressed the growth of thyroid cancer cells and tumor xenografts via inhibiting PI3K/Akt/mTOR and CPSF4/hTERT signaling and reactivating mutant p53.

mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing.

Pre-mRNA alternative splicing and alternative polyadenylation have been implicated to play important roles during eukaryotic gene expression. However, much remains unknown regarding the regulatory mechanisms and the interactions of these two processes in plants. Here we focus on an Arabidopsis gene OXT6 (Oxidative Tolerant-6) that has been demonstrated to encode two proteins through alternative splicing and alternative polyadenylation. Specifically, alternative polyadenylation at Intron-2 of OXT6 produces a transcript coding for AtCPSF30, an Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. On the other hand, alternative splicing of Intron-2 generates a longer transcript encoding a protein named AtC30Y, a polypeptide including most part of AtCPSF30 and a YT521B domain. To investigate the expression outcome of OXT6 in plants, a set of mutations were constructed to alter the splicing and polyadenylation patterns of OXT6. Analysis of transgenic plants bearing these mutations by quantitative RT-PCR revealed a competition relationship between these two processes. Moreover, when both splice sites and poly(A) signals were mutated, polyadenylation became the preferred mode of OXT6 processing. These results demonstrate the interplay between alternative splicing and alternative polyadenylation, and it is their concerted actions that define a gene’s expression outcome.

Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii . Parasites selected to be resistant to AN3661 had mutations in TgCPSF3 , which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF‐73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild‐type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, Tg CPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti‐parasitic drugs.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood. Our recent findings demonstrate that alternative polyadenylation (APA) caused by NUDT21 reduction is important for the increased expression of fibrotic mediators and ECM proteins in lung fibroblasts by shortening the 3′-untranslated regions (3′-UTRs) of mRNAs and stabilizing their transcripts, therefore activating pathological signaling pathways. Despite the importance of NUDT21 reduction in the regulation of fibrosis, the underlying mechanisms for the depletion are unknown. We demonstrate here that NUDT21 is depleted by TGFβ1. We found that miR203, which is increased in IPF, was induced by TGFβ1 to target the NUDT21 3′-UTR, thus depleting NUDT21 in human and mouse lung fibroblasts. TGFβ1-mediated NUDT21 reduction was attenuated by the miR203 inhibitor antagomiR203 in fibroblasts. TGFβ1 transgenic mice revealed that TGFβ1 down-regulates NUDT21 in fibroblasts in vivo. Furthermore, TGFβ1 promoted differential APA of fibrotic genes, including FGF14, RICTOR, TMOD2, and UCP5, in association with increased protein expression. This unique differential APA signature was also observed in IPF fibroblasts. Altogether, our results identified TGFβ1 as an APA regulator through NUDT21 depletion amplifying pulmonary fibrosis.

Pre-mRNA 3'-end maturation is achieved by a mechanism requiring four different protein complexes assembled from approximately twenty factors. A global understanding of this essential process is still missing due to the inability to structurally characterize the entire complexes, even though structures of the isolated factors have been obtained. In this review, we summarize recent findings regarding the atomic description of one of the major players, the Cleavage and Polyadenylation Specificity Factor complex (CPSF in human, CPF in yeast). These data provide information on the architecture adopted by the major components of this complex, and on its capacity to recognize the polyadenylation signal sequence.

A crucial step for mRNA polyadenylation is poly(A) signal recognition by trans-acting factors. The mammalian cleavage and polyadenylation specificity factor (CPSF) complex components CPSF30 and WD repeat-containing protein33 (WDR33) recognize the canonical AAUAAA for polyadenylation. In Arabidopsis (Arabidopsis thaliana), the flowering time regulator FY is the homolog of WDR33. However, its role in mRNA polyadenylation is poorly understood. Using poly(A) tag sequencing, we found that >50% of alternative polyadenylation (APA) events are altered in fy single mutants or double mutants with oxt6 (a null mutant of AtCPSF30), but mutation of the FY WD40-repeat has a stronger effect than deletion of the plant-unique Pro-Pro-Leu-Pro-Pro (PPLPP) domain. fy mutations disrupt AAUAAA or AAUAAA-like poly(A) signal recognition. Notably, A-rich signal usage is suppressed in the WD40-repeat mutation but promoted in PPLPP-domain deficiency. However, fy mutations do not aggravate the altered signal usage in oxt6. Furthermore, the WD40-repeat mutation shows a preference for 3′ untranslated region shortening, but the PPLPP-domain deficiency shows a preference for lengthening. Interestingly, the WD40-repeat mutant exhibits shortened primary roots and late flowering with alteration of APA of related genes. Importantly, the long transcripts of two APA genes affected in fy are related to abiotic stress responses. These results reveal a conserved and specific role of FY in mRNA polyadenylation.

Solving the structure of large, multi-subunit complexes is difficult despite recent advances in cryoEM, due to remaining challenges to express and purify complex subunits. Computational approaches that predict protein-protein interactions, including Direct Coupling Analysis (DCA), represent an attractive alternative to dissect interactions within protein complexes. However, due to high computational complexity and high false positive rate they are applicable only to small proteins. Here, we present a modified DCA to predict residues and domains involved in interactions of large proteins. To reduce false positive levels and increase accuracy of prediction, we use local Gaussian averaging and predicted secondary structure elements. As a proof-of-concept, we apply our method to two Integrator subunits, INTS9 and INTS11, which form a heterodimeric structure previously solved by crystallography. We accurately predict the domains of INTS9/11 interaction. We then apply this approach to predict the interaction domains of two complexes whose structure is currently unknown: 1) The heterodimer formed by the Cleavage and Polyadenylation Specificity Factor 100-kD (CPSF100) and 73-kD (CPSF73); 2) The heterotrimer formed by INTS4/9/11. Our predictions of interactions within these two complexes are supported by experimental data, demonstrating that our modified DCA is a useful method for predicting interactions and can easily be applied to other complexes.

Summary Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPFcore” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site., Graphical Abstract, Highlights • Crystallography and cryo-EM reveal a Ysh1-Mpe1 interface • Specific pre-mRNA 3ʹ end cleavage is reconstituted from recombinant proteins • Electron microscopy shows that CPFcore assembles around a central scaffold • A model for activation of the 3ʹ endonuclease on substrate RNAs is proposed, The 3ʹ ends of eukaryotic mRNAs are formed by endonucleolytic cleavage by Ysh1/CPSF73. Hill et al. define a minimal machinery for cleavage and polyadenylation in vitro using recombinant yeast proteins and short substrates. They elucidate the architecture of an ∼500 kDa eight-protein assembly, including the atomic details of a Ysh1-Mpe1 interface.

Alternative polyadenylation (APA) is how genes choose different sites for 3' end formation for mRNAs during transcription. APA often occurs in a tissue- or developmental stage-specific manner that can significantly affect gene activity by changing the protein product generated, the stability of the transcript, its localization within the cell, or its translatability. Despite the important regulatory effects that APA has on tissue-specific gene expression, only a few examples have been characterized mechanistically. In this 2018 update to our 2010 review, we examine mechanisms for the control of APA and update our understanding of the older mechanisms since 2010. We once postulated the existence of tissue-specific factors in APA. However, while a few tissue-specific polyadenylation factors are known, the emerging conclusion is that the majority of APA is accomplished by altering levels of core polyadenylation proteins. Examples of those core proteins include CSTF2, CPSF1, and subunits of mammalian cleavage factor I. But despite support for these mechanisms, no one has yet documented any of these proteins changing in either a tissue-specific or developmental manner. Given the profound effect that APA can have on gene expression and human health, improved understanding of tissue-specific APA could lead to numerous advances in gene activity control. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Development.

International audience; Cryptosporidium is an intestinal pathogen that causes severe but self-limiting diarrhea in healthy humans, yet it can turn into a life-threatening, unrelenting infection in immunocompromised patients and young children. Severe diarrhea is recognized as the leading cause of mortality for children below 5 years of age in developing countries. The only approved treatment against cryptosporidiosis, nitazoxanide, has limited efficacy in the most vulnerable patient populations, including malnourished children, and is ineffective in immunocompromised individuals. Here, we investigate inhibition of the parasitic cleavage and polyadenylation specificity factor 3 (CPSF3) as a strategy to control Cryptosporidium infection. We show that the oxaborole AN3661 selectively blocked Cryptosporidium growth in human HCT-8 cells, and oral treatment with AN3661 reduced intestinal parasite burden in both immuno-compromised and neonatal mouse models of infection with greater efficacy than nitazoxanide. Furthermore, we present crystal structures of recombinantly produced Cryptosporidium CPSF3, revealing a mechanism of action whereby the mRNA processing activity of this enzyme is efficiently blocked by the binding of the oxaborole group at the metal-dependent catalytic center. Our data provide insights that may help accelerate the development of next-generation anti-Cryptosporidium therapeutics.

Broad Copy Number Gains (BCNGs) are copy-number increases of chromosomes or large segments of chromosomal arms. Publicly-available single-nucleotide polymorphism (SNP) array and RNA-Seq data of colon adenocarcinoma (COAD) samples from The Cancer Genome Atlas (TCGA) consortium allowed us to design better control groups in order to identify changes in expression due to highly recurrent BCNGs (in chromosomes 20, 8, 7, 13). We identified: (1) Overexpressed Transcripts (OverT), transcripts whose expression increases in &ldquo, COAD groups bearing a specific BCNG&rdquo, in comparison to &ldquo, control COAD groups&rdquo, not bearing it, and (2) up-regulated/down-regulated transcripts, transcripts whose expression increases/decreases in COAD groups in comparison to normal colon tissue. An analysis of gene expression reveals a correlation between the density of up-regulated genes per selected chromosome and the recurrence rate of their BCNGs. We report an enrichment of gained enhancer activity and of cancer fitness genes among OverT genes. These results support the hypothesis that the chromosomal density of overexpressed cancer fitness genes might play a significant role in the selection of gained chromosomes during cancer evolution. Analysis of functional pathways associated with OverT suggest that some multi-subunit protein complexes (eIF2, eIF3, CSTF and CPSF) are candidate targets for silencing transcriptional therapy.

Most human genes have multiple sites at which RNA 3' end cleavage and polyadenylation can occur, enabling the expression of distinct transcript isoforms under different conditions. Novel methods to sequence RNA 3' ends have generated comprehensive catalogues of polyadenylation (poly(A)) sites; their analysis using innovative computational methods has revealed how poly(A) site choice is regulated by core RNA 3' end processing factors, such as cleavage factor I and cleavage and polyadenylation specificity factor, as well as by other RNA-binding proteins, particularly splicing factors. Here, we review the experimental and computational methods that have enabled the global mapping of mRNA and of long non-coding RNA 3' ends, quantification of the resulting isoforms and the discovery of regulators of alternative cleavage and polyadenylation (APA). We highlight the different types of APA-derived isoforms and their functional differences, and illustrate how APA contributes to human diseases, including cancer and haematological, immunological and neurological diseases. published

Global, segmental, and gene duplication–related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)–assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana. Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3′-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.

Correct 3’end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

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.

Objectives Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is a highly regulated process. Most human genes have multiple poly(A) sites, which define different possible mRNA ends, suggesting the existence of mechanisms that regulate which poly(A) site is used. Poly(A) site selection may be mediated by cleavage factor I (CFIm), which is part of the cleavage and polyadenylation (CPA) complex. CFIm comprises CFIm25, CFIm59 and CFim68 subunits. It has been documented that the CPA complex also regulates pol II transcription at the start of genes. We therefore investigated whether CFIm, in addition to its role in poly(A) site selection, is involved in the regulation of pol II transcription. Data description We provide genome-wide data of the effect of reducing by 90% expression of the CFIm25 constituent of CFIm, which is involved in pre-mRNA cleavage and polyadenylation, on pol II transcription in human cells. We performed pol II ChIP-seq in the presence or absence of CFIm25 and with or without an inhibitor of the cyclin-dependent kinase (CDK)9, which regulates the entry of pol II into productive elongation.

H9N2 influenza A viruses (IAV) are considered low pathogenic avian influenza viruses (LPAIV). These viruses are endemic in poultry in many countries in Asia, the Middle East and parts of Africa. Several cases of H9N2-associated infections in humans as well as in pigs have led the World Health Organization (WHO) to include these viruses among those with pandemic potential. To date, the processes and mechanisms associated with H9N2 IAV adaptation to mammals are poorly understood. The non-structural protein 1 (NS1) from IAV is a virulence factor that counteracts the innate immune responses. Here, we evaluated the ability of the NS1 protein from A/quail/Hong Kong/G1/97 (HK/97) H9N2 to inhibit host immune responses. We found that HK/97 NS1 protein counteracted interferon (IFN) responses but was not able to inhibit host gene expression in human or avian cells. In contrast, the NS1 protein from earlier H9N2 IAV strains, including the first H9N2 A/turkey/Wisconsin/1/1966 (WI/66), were able to inhibit both IFN and host gene expression. Using chimeric constructs between WI/66 and HK/97 NS1 proteins, we identified the region and amino acid residues involved in inhibition of host gene expression. Amino acid substitutions L103F, I106M, P114S, G125D and N139D in HK/97 NS1 resulted in binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) and, in consequence, inhibition of host gene expression. Notably, changes in the same amino acid residues resulted in the lack of inhibition of host gene expression by WI/66 NS1. Importantly, our results identified a new combination of amino acids required for NS1 binding to CPSF30 and inhibition of host gene expression. These results also confirm previous studies demonstrating strain specific differences in the ability of NS1 proteins to inhibit host gene expression.

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization’s target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei . Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

The influenza virus is a significant public health concern causing 250,000–500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1(ED)) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918(H1N1)) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918(H1N1) NS1(ED). Furthermore, we mapped the A9/A22-binding site onto our 1918(H1N1) NS1(ED) structure and determined that A9 and A22 interact with the NS1(ED) in the hydrophobic pocket known to facilitate binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1(ED)–CPSF30 interaction. Finally, our structure revealed that NS1(ED) could dimerize via an interface that we termed the α(3)–α(3) dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro -assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358. IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo . Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.