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The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process. Given the importance of alternative cleavage and polyadenylation in increasing the diversity of mRNAs, the exact mechanism of binding of RNA to the RRM of CSTF2 remains unclear, particularly in the absence of a structure of this RRM bound to a native RNA substrate. Here, we performed a series of NMR titration and spin relaxation experiments, which were complemented by paramagnetic relaxation enhancement measurements and rigid-body docking, to characterize the interactions of the CSTF2 RRM with a U-rich ligand. Our results reveal a multistep binding process involving differences in ps-ns time scale dynamics and potential structural changes, particularly in the C-terminalα-helix. These results provide insights into how the CSTF2 RRM domain binds to U-rich RNA ligands and offer a greater understanding for the molecular basis of the regulation of pre-mRNA cleavage and polyadenylation.

A protein called cleavage-stimulating factor subunit 2 (CSTF2, additionally called CSTF-64) binds RNA and is needed for the cleavage and polyadenylation of mRNA. CSTF2 is an important component subunit of the cleavage stimulating factor (CSTF), which is located on the X chromosome and encodes 557 amino acids. There is compelling evidence linking elevated CSTF2 expression to the pathological advancement of cancer and on its impact on the clinical aspects of the disease. The progression of cancers, including hepatocellular carcinoma, melanoma, prostate cancer, breast cancer, and pancreatic cancer, is correlated with the upregulation of CSTF2 expression. This review provides a fresh perspective on the investigation of the associations between CSTF2 and various malignancies and highlights current studies on the regulation of CSTF2. In particular, the mechanism of action and potential clinical applications of CSTF2 in cancer suggest that CSTF2 can serve as a new biomarker and individualized treatment target for a variety of cancer types.

Heat shock protein 101 (HSP101) in plants, and bacterial and yeast orthologs, is essential for thermotolerance. To investigate thermotolerance mechanisms involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a missense HSP101 allele (hot1-4). hot1-4 plants are sensitive to acclimation heat treatments that are otherwise permissive for HSP101 null mutants, indicating that the hot1-4 protein is toxic. We report one suppressor (shot2, suppressor of hot1-4 2) has a missense mutation of a conserved residue in CLEAVAGE STIMULATION FACTOR77 (CstF77), a subunit of the polyadenylation complex critical for mRNA 3' end maturation. We performed ribosomal RNA depletion RNA-Seq and captured transcriptional readthrough with a custom bioinformatics pipeline. Acclimation heat treatment caused transcriptional readthrough in hot1-4 shot2, with more readthrough in heat-induced genes, reducing the levels of toxic hot1-4 protein and suppressing hot1-4 heat sensitivity. Although shot2 mutants develop like the wild type in the absence of stress and survive mild heat stress, reduction of heat-induced genes and decreased HSP accumulation makes shot2 in HSP101 null and wild-type backgrounds sensitive to severe heat stress. Our study reveals the critical function of CstF77 for 3' end formation of mRNA and the dominant role of HSP101 in dictating the outcome of severe heat stress., Competing Interests: Conflict of interest statement. The authors declare no competing interests., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Effective therapies for renal fibrosis, the common endpoint for most kidney diseases, are lacking. We previously reported that alternative polyadenylation (APA) drives transition from acute kidney injury to chronic kidney disease, suggesting a potential role for APA in renal fibrogenesis. Here, we found that among canonical APA writers, CSTF2 expression was upregulated in tubular epithelial cells (TEC) of fibrotic kidneys. CSTF2 was also identified as a TGF-β-inducible pro-fibrotic gene. Further analysis revealed that CSTF2 promoted epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) overproduction in TEC by inducing 3'UTR shortening and upregulation of the expression of basic fibroblast growth factor 2 (FGF2). Additionally, 3'UTR shortening stabilised FGF2 mRNA through miRNA evasion. Interestingly, FGF2 enhanced CSTF2 expression, leading to the forming of a CSTF2-FGF2 positive loop in TEC. Furthermore, CSTF2 knockdown alleviated unilateral ureteral obstruction-induced renal fibrosis in vivo. Finally, we developed a CSTF2-targeted antisense oligonucleotide (ASO) and validated its effectiveness in vitro. These results indicate that the expression of the APA writer, CSTF2, is upregulated by TGF-β and CSTF2 facilitates TGF-β-induced FGF2 overexpression, forming a TGF-β-CSTF2-FGF2 pro-fibrotic axis in TEC. CSTF2 is a potentially promising target for renal fibrosis that does not directly disrupt TGF-β., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

3' end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3' end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal 'acidic' region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3' polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3' end processing., Competing Interests: LM, AM, FB, MC, MJ No competing interests declared, (© 2022, Muckenfuss et al.)

The majority of human genes have multiple polyadenylation sites, which are differentially used through the process of alternative polyadenylation (APA). Dysregulation of APA contributes to numerous diseases, including cancer. However, specific genes subject to APA that impact oncogenesis have not been well characterized, and many cancer APA landscapes remain underexplored. Here, we used dynamic analyses of APA from RNA-seq (DaPars) to define both the 3'UTR APA profile in esophageal squamous cell carcinoma (ESCC) and to identify 3'UTR shortening events that may drive tumor progression. In four distinct squamous cell carcinoma datasets, BID 3'UTRs were recurrently shortened and BID mRNA levels were significantly upregulated. Moreover, system correlation analysis revealed that CstF64 is a candidate upstream regulator of BID 3'UTR length. Mechanistically, a shortened BID 3'UTR promoted proliferation of ESCC cells by disrupting competing endogenous RNA (ceRNA) cross-talk, resulting in downregulation of the tumor suppressor gene ZFP36L2 . These in vitro and in vivo results were supported by human patient data whereby 3'UTR shortening of BID and low expression of ZFP36L2 are prognostic factors of survival in ESCC. Collectively, these findings demonstrate that a key ceRNA network is disrupted through APA and promotes ESCC tumor progression. Significance: High-throughput analysis of alternative polyadenylation in esophageal squamous cell carcinoma identifies recurrent shortening of the BID 3'UTR as a driver of disease progression., (©2021 American Association for Cancer Research.)

CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the significance of 3' end mRNA processing in expression of genes important for brain plasticity and neuronal development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression.

Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3' end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1-CPAFs), that binds intronic PASs and suppresses PCPA. U1-CPAFs are distinct from U1-spliceosomal complexes; they include CPA's three main subunits, CFIm, CPSF, and CstF; lack essential splicing factors; and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1-CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1-CPAFs' switch from repressive to activated states. Our findings outline this U1 telescripting mechanism and demonstrate U1's unique role as central regulator of pre-mRNA processing and transcription., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Polyadenylation influences gene expression by affecting mRNA stability, transport, and translatability. Here, we report that Cleavage stimulation Factor 77 (AtCstF77), a component of the pre-mRNA 3'-end polyadenylation machinery, affects polyadenylation site (PAS) selection in transcripts of some auxin signaling genes in Arabidopsis ( Arabidopsis thaliana ). Disruption of AtCstF77 reduced auxin sensitivity and decreased the expression of the auxin reporter DR5 - GFP Null mutations of cstf77 caused severe developmental defects, but were not lethal as previously reported. cstf77 - 2 genetically interacted with transport inhibitor response 1 auxin signaling f - box 2 auxin receptor double mutants, further supporting that polyadenylation affects auxin signaling. AtCstF77 was ubiquitously expressed in embryos, seedlings, and adult plants. The AtCstF77 protein was localized in the nucleus, which is consistent with its function in pre-mRNA processing. We observed that PASs in transcripts from approximately 2,400 genes were shifted in the cstf77 - 2 mutant. Moreover, most of the PAS shifts were from proximal to distal sites. Auxin treatment also caused PAS shifts in transcripts from a small number of genes. Several auxin signaling or homeostasis genes had different PASs in their transcripts in the cstf77 - 2 mutant. The expression levels of AUXIN RESISTANT 2 / INDOLE - 3 - ACETIC ACID 7 were significantly increased in the cstf77 - 2 mutant, which can partially account for the auxin resistance phenotype of this mutant. Our results demonstrate that AtCstF77 plays pleiotropic and critical roles in Arabidopsis development. Moreover, disruption of AtCstF64, another component of the polyadenylation machinery, led to developmental defects and reduced auxin response, similar to those of the cstf77 - 2 mutant. We conclude that AtCstF77 affects auxin responses, likely by controlling PAS selection of transcripts of some auxin signaling components., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR., (Copyright © 2018 Fonseca et al.)

Cleavage stimulation factor (CstF) is a highly conserved protein complex composed of three subunits that recognizes G/U-rich sequences downstream of the polyadenylation signal of eukaryotic mRNAs. While CstF has been identified over 25 years ago, the architecture and contribution of each subunit to RNA recognition have not been fully understood. In this study, we provide a structural basis for the recruitment of CstF-50 to CstF via interaction with CstF-77 and establish that the hexameric assembly of CstF creates a high affinity platform to target various G/U-rich sequences. We further demonstrate that CstF-77 boosts the affinity of the CstF-64 RRM to the RNA targets and CstF-50 fine tunes the ability of the complex to recognize G/U sequences of certain lengths and content., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5' end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3' end of the mRNA. Furthermore, the 3' processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5' and the 3' regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3' processing., (© Crown copyright 2017. This article contains public sector information licensed under the Open Government Licence v3.0 (http://www.nationalarchives.gov.uk/doc/open-governmentlicence/version/3/).)

Polyadenylation is an essential mechanism for the processing of mRNA 3' ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNA-binding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction., Competing Interests: The authors have declared that no competing interests exist.

Spermatogenesis is coordinated by the spatial and temporal expression of many transcriptional and posttranscriptional factors. The cyclic AMP-responsive element modulator (CREM) gene encodes both activator and repressor isoforms that act as transcription factors to regulate spermiogenesis. We found that the testis-expressed paralog of CstF-64, tauCstF-64 (gene symbol Cstf2t), is involved in a polyadenylation site choice switch of Crem mRNA and leads to an overall decrease of the Crem mRNAs that are generated from internal promoters in Cstf2t(-/-) mice. More surprisingly, loss of tauCstF-64 also leads to alternative splicing of Crem exon 4, which contains an important activation domain. Thus, testis-specific CREMtau2 isoform protein levels are reduced in Cstf2t(-/-) mice. Consequently, expression of 15 CREM-regulated genes is decreased in testes of Cstf2t(-/-) mice at 25 days postpartum. These effects might further contribute to the infertility phenotype of these animals. This demonstrates that tauCstF-64 is an important stage-specific regulator of Crem mRNA processing that modulates the spatial and temporal expression of downstream stage-specific genes necessary for the proper development of sperm in mice., (© 2016 by the Society for the Study of Reproduction, Inc.)

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Although adult cardiomyocytes have the capacity for cellular regeneration, they are unable to fully repair severely injured hearts. The use of embryonic stem cell (ESC)-derived cardiomyocytes as transplantable heart muscle cells has been proposed as a solution, but is limited by the lack of understanding of the developmental pathways leading to specification of cardiac progenitors. Identification of these pathways will enhance the ability to differentiate cardiomyocytes into a clinical source of transplantable cells. Here, we show that the mRNA 3' end processing protein, CstF-64, is essential for cardiomyocyte differentiation in mouse ESCs. Loss of CstF-64 in mouse ESCs results in loss of differentiation potential toward the endodermal lineage. However, CstF-64 knockout (Cstf2(E6)) cells were able to differentiate into neuronal progenitors, demonstrating that some differentiation pathways were still intact. Markers for mesodermal differentiation were also present, although Cstf2(E6) cells were defective in forming beating cardiomyocytes and expressing cardiac specific markers. Since the extraembryonic endoderm is needed for cardiomyocyte differentiation and endodermal markers were decreased, we hypothesized that endodermal factors were required for efficient cardiomyocyte formation in the Cstf2(E6) cells. Using conditioned medium from the extraembryonic endodermal (XEN) stem cell line we were able to restore cardiomyocyte differentiation in Cstf2(E6) cells, suggesting that CstF-64 has a role in regulating endoderm differentiation that is necessary for cardiac specification and that extraembryonic endoderm signaling is essential for cardiomyocyte development., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G1 phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G1 phase, although the details of this mechanism are not well understood. Similarly, the role of 3' end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3' end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G1 phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3' end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3' end processing., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.

Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system., (© 2013 Elsevier B.V. All rights reserved.)

The human gene encoding the cleavage/polyadenylation (C/P) factor CstF-77 contains 21 exons. However, intron 3 (In3) accounts for nearly half of the gene region, and contains a C/P site (pA) with medium strength, leading to short mRNA isoforms with no apparent protein products. This intron contains a weak 5' splice site (5'SS), opposite to the general trend for large introns in the human genome. Importantly, the intron size and strengths of 5'SS and pA are all highly conserved across vertebrates, and perturbation of these parameters drastically alters intronic C/P. We found that the usage of In3 pA is responsive to the expression level of CstF-77 as well as several other C/P factors, indicating it attenuates the expression of CstF-77 via a negative feedback mechanism. Significantly, intronic C/P of CstF-77 pre-mRNA correlates with global 3'UTR length across cells and tissues. In addition, inhibition of U1 snRNP also leads to regulation of the usage of In3 pA, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes. Importantly, perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation. Thus, the conserved intronic pA of the CstF-77 gene may function as a sensor for cellular C/P and splicing activities, controlling the homeostasis of CstF-77 and C/P activity and impacting cell proliferation and differentiation., Competing Interests: The authors declare competing financial interests: B. Tian, M. Hogue, Z. Ji, and W. Luo are named on the pending US patent application no. PCT/US2012/052122 based on this work.

The general transcription factor II B (TFIIB) plays a central role in both the assembly of the transcription complex at gene promoters and also in the events that lead to transcription initiation. TFIIB is phosphorylated at serine-65 at the promoters of several endogenous genes, and this modification is required to drive the formation of gene promoter-3' processing site contacts through the cleavage stimulation factor 3' (CstF 3')-processing complex. Here we demonstrate that TFIIB phosphorylation is dispensable for the transcription of genes activated by the p53 tumor suppressor. We find that the kinase activity of TFIIH is critical for the phosphorylation of TFIIB serine-65, but it is also dispensable for the transcriptional activation of p53-target genes. Moreover, we demonstrate that p53 directly interacts with CstF independent of TFIIB phosphorylation, providing an alternative route to the recruitment of 3'-processing complexes to the gene promoter. Finally, we show that DNA damage leads to a reduction in the level of phospho-ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. Our data reveal a mode of phospho-TFIIB-independent transcriptional regulation that prioritizes the transcription of p53-target genes during cellular stress.

The τCstF-64 polyadenylation protein (gene symbol Cstf2t) is a testis-expressed orthologue of CstF-64. Mice in which Cstf2t was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and Cstf2t(-/-) mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of Cstf2t(-/-) mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in Cstf2t(-/-) testes. These results suggest that τCstF-64 plays a role in 3' end determination and transcription termination for a large range of germ cell-expressed genes.

3'-end cleavage and subsequent polyadenylation are critical steps in mRNA maturation. The precise location where cleavage occurs (referred to as poly(A) site) is determined by a tripartite mechanism in which a A(A/U)UAAA hexamer, GU rich downstream element and UGUA upstream element are recognized by the cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CstF) and cleavage factor I(m) (CFI(m)), respectively. CFI(m) is composed of a smaller 25 kDa subunit (CFI(m)25) and a larger 59, 68 or 72 kDa subunit. CFI(m)68 interacts with CFI(m)25 through its N-terminal RNA recognition motif (RRM). We recently solved the crystal structures of CFI(m)25 bound to RNA and of a complex of CFI(m)25, the RRM domain of CFI(m)68 and RNA. Our study illustrated the molecular basis for UGUA recognition by the CFI(m) complex, suggested a possible mechanism for CFI(m) mediated alternative polyadenylation, and revealed potential links between CFI(m) and other mRNA processing factors, such as the 20 kDa subunit of the cap binding protein (CBP20), and the splicing regulator U2AF65.

Cleavage/polyadenylation of mRNAs and 3' processing of replication-dependent histone transcripts are both mediated by large complexes that share several protein components. Functional studies of these shared proteins are complicated by the cooperative binding of the individual subunits. For CstF-64, an additional difficulty is that symplekin and CstF-77 bind mutually exclusively to its hinge domain. Here we have identified CstF-64 and symplekin mutants that allowed us to distinguish between these interactions and to elucidate the role of CstF-64 in the two processing reactions. The interaction of CstF-64 with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation. In contrast, the nuclear accumulation of CstF-64 depends on its binding to CstF-77 and not to symplekin. Moreover, the CstF-64 paralogue CstF-64Tau can compensate for the loss of CstF-64. As CstF-64Tau has a lower affinity for CstF-77 than CstF-64 and is relatively unstable, it is the minor form. However, it may become up-regulated when the CstF-64 level decreases, which has biological implications for spermatogenesis and probably also for other regulatory events. Thus, the interactions between CstF-64/CstF-64Tau and CstF-77 are important for the maintenance of stoichiometric nuclear levels of the CstF complex components and for their intracellular localization, stability, and function.

Fertilization is a multistep process requiring spermatozoa with unique cellular structures and numerous germ cell-specific molecules that function in the various steps. In the highly coordinated process of male germ cell development, RNA splicing and polyadenylation help regulate gene expression to assure formation of functional spermatozoa. Male germ cells express tauCstF-64 (Cstf2t gene product), a paralog of the X-linked CstF-64 protein that supports polyadenylation in most somatic cells. We previously showed that loss of tauCstF-64 causes male infertility because of major defects in mouse spermatogenesis. Surprisingly, although Cstf2t(-/-) males produce very few recognizable spermatozoa, some of the spermatozoa produced are motile. This led us to ask whether these Cstf2t(-/-) sperm were fertile. A motile cell-enriched population of spermatozoa from Cstf2t-null males dispersed cumulus cells of cumulus-oocyte complexes normally. However, motile spermatozoa from Cstf2t-null males failed to fertilize cumulus-intact mouse eggs in vitro. In addition, sperm adhesion to the zona pellucida (ZP) of cumulus-free eggs was significantly decreased, indicating tauCstF-64 is required for production of spermatozoa capable of ZP interaction. Acrosomal proteins involved in sperm-ZP recognition, including zonadhesin, proacrosin, SPAM1/PH-20, and ZP3R/sp56, were normally distributed in the apical head of Cstf2t(-/-) spermatozoa. We conclude that tauCstF-64 is required not only for expression of genes involved in morphological differentiation of spermatids but also for genes having products that function during interaction of motile spermatozoa with eggs. To our knowledge, this is the first demonstration that a gene involved in polyadenylation has a negative consequence on sperm-ZP adhesion.

Noncoding RNA is emerging as an important regulator of gene expression in many organisms. We are characterizing RNA-mediated chromatin silencing of the Arabidopsis major floral repressor gene, FLC. Through suppressor mutagenesis, we identify a requirement for CstF64 and CstF77, two conserved RNA 3'-end-processing factors, in FLC silencing. However, FLC sense transcript 3' processing is not affected in the mutants. Instead, CstF64 and CstF77 are required for 3' processing of FLC antisense transcripts. A specific RNA-binding protein directs their activity to a proximal antisense polyadenylation site. This targeted processing triggers localized histone demethylase activity and results in reduced FLC sense transcription. Targeted 3' processing of antisense transcripts may be a common mechanism triggering transcriptional silencing of the corresponding sense gene.

Immunoglobulin secretion is modulated by competition between the use of a weak promoter-proximal poly(A) site and a nonconsensus splice site in the final secretory-specific exon of the heavy chain pre-mRNA. The RNA polymerase II transcription elongation factor ELL2, which is induced in plasma cells, enhanced both polyadenylation and exon skipping with the gene encoding the immunoglobulin heavy-chain complex (Igh) and reporter constructs. Lowering ELL2 expression by transfection of heterogenous ribonucleoprotein F (hnRNP F) or small interfering RNA resulted in lower abundance of secretory-specific forms of immunoglobulin heavy-chain mRNA. ELL2 and the polyadenylation factor CstF-64 tracked together with RNA polymerase II across the Igh mu- and gamma-gene segments; the association of both factors was blocked by ELL2-specific small interfering RNA. Thus, loading of ELL2 and CstF-64 on RNA polymerase II was linked, caused enhanced use of the proximal poly(A) site and was necessary for processing of immunoglobulin heavy-chain mRNA.

Metazoan histone mRNAs are unique: their pre-mRNAs contain no introns, and the mRNAs are not polyadenylated, ending instead in a conserved stem-loop structure. In Drosophila, canonical poly(A) signals are located downstream of the normal cleavage site of each histone gene and are utilized when histone 3' end formation is inhibited. Here we define a subcomplex of poly(A) factors that are required for histone pre-mRNA processing. We demonstrate that Symplekin, CPSF73, and CPSF100 are present in a stable complex and interact with histone-specific processing factors. We use chromatin immunoprecipitation to show that Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes. Depletion of SLBP recruits CstF50 to histone genes. Knockdown of CPSF160 or CstF64 downregulates Symplekin but does not affect histone pre-mRNA processing or association of Symplekin with the histone locus. These results suggest that a common core cleavage factor is required for processing of histone and polyadenylated pre-mRNAs.

Background: Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs., Results: We discovered several closely related splice variants of CstF-64 - collectively called betaCstF-64 - that could potentially contribute to proteomic diversity in the nervous system. The betaCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major betaCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of betaCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of betaCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to betaCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since betaCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that betaCstF-64 has an evolutionarily conserved function in these animals. betaCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for betaCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that betaCstF-64 is expressed in neurons but not glia., Conclusion: This is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that betaCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs.

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

RNA polymerase II (Pol II) transcription termination involves two linked processes: mRNA 3'-end formation and release of Pol II from DNA. Signals for 3' processing are recognized by a protein complex that includes cleavage polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF). Here we identify suppressors encoding proteins that play roles in processes at the 3' ends of genes by exploiting a mutation in which the 3' end of another gene is transposed into the first gene of the Caenorhabditis elegans lin-15 operon. As expected, genes encoding CPSF and CstF were identified in the screen. We also report three suppressors encoding proteins containing a domain that interacts with the C-terminal domain of Pol II (CID). We show that two of the CID proteins are needed for efficient 3' cleavage and thus may connect transcription termination with RNA cleavage. Furthermore, our results implicate a serine/arginine-rich (SR) protein, SRp20, in events following 3'-end cleavage, leading to termination of transcription.

In addition to the proteolytic cleavages that give rise to amyloid-beta (Abeta), the amyloid-beta protein precursor (AbetaPP) is cleaved at Asp664 intracytoplasmically. This cleavage releases a cytotoxic peptide, APP-C31, removes AbetaPP-interaction motifs required for signaling and internalization, and is required for the generation of AD-like deficits in a mouse model of the disease. Although we and others had previously shown that Asp664 cleavage of AbetaPP is increased in AD brains, the distribution of the Asp664-cleaved forms of AbetaPP in non-diseased and AD brains at different ages had not been determined. Confirming previous reports, we found that Asp664-cleaved forms of AbetaPP were increased in neuronal cytoplasm and nuclei in early-stage AD brains but were absent in age-matched, non-diseased control brains and in late-stage AD brains. Remarkably, however, Asp664-cleaved AbetaPP was prominent in neuronal somata and in processes in entorhinal cortex and hippocampus of non-diseased human brains at ages <45 years. Our observations suggest that Asp664 cleavage of AbetaPP may be part of the normal proteolytic processing of AbetaPP in young (<45 years) human brain and that this cleavage is down-regulated with normal aging, but is aberrantly increased and altered in location in early AD.

Polyadenylation, the process of eukaryotic mRNA 3' end formation, is essential for gene expression and cell viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, tauCstF-64 (gene name Cstf2t), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for tauCstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of Cstf2t(-/-) mice as measured by microarray. Our results indicate that, although the infertility in Cstf2t(-/-) males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on tauCstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes.

The cleavage-stimulation factor (CstF) is required for the cleavage of the 3'-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality.

To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV-TCR). Using nuclear extracts from the JCV-susceptible neuroblastoma cell line IMR-32, EMSA revealed a 670 kDa JCV-TCR-binding protein complex designated as #3-bp. This complex could not be detected in nuclear extracts from non-susceptible cell lines. Using column chromatographic purifi-cation and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3-bp. However, as CstF is present in many cell types, we speculated that the IMR-32-specific component(s) of #3-bp bind CstF. We performed a yeast two-hybrid assay using CstF-77 as the bait against a HeLa cDNA-subtracted IMR-32 cDNA library. This analysis detected binding between CstF-77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV-TCR. Our findings indicate that an association between DDX1 and the JCV-TCR may play a significant role in JCV infection in IMR-32 cells.

Recently, we demonstrated that the DEAD box protein 1 (DDX1), an RNA helicase, and the cleavage stimulation factor (CstF) form a complex that binds to the JC virus transcriptional control region (JCV-TCR). Here, we examined the function of DDX1, which is expressed at much higher levels in the JCV-susceptible cell line IMR-32 than in non-susceptible cell lines. DDX1 had no effect on the replication efficiency of JCV, but overexpression of DDX1 significantly increased transactivation of the JCV promoter. Furthermore, DDX1 enhanced the expression of JCV proteins in JCV infected cells, and knockdown of DDX1 using small interfering (si) RNA suppressed the expression of JCV proteins. Our results clearly demonstrate that DDX1 regulates proliferation of JCV in vitro through transcriptional activation.

Many eukaryotic cells use RNA-directed silencing mechanisms to protect against viruses and transposons and to suppress endogenous gene expression at the posttranscriptional level. RNA silencing also is implicated in epigenetic mechanisms affecting chromosome structure and transcriptional gene silencing. Here, we describe enhanced silencing phenotype (esp) mutants in Arabidopsis thaliana that reveal how proteins associated with RNA processing and 3' end formation can influence RNA silencing. These proteins were a putative DEAH RNA helicase homologue of the yeast PRP2 RNA splicing cofactor and homologues of mRNA 3' end formation proteins CstF64, symplekin/PTA1, and CPSF100. The last two proteins physically associated with the flowering time regulator FY in the 3' end formation complex AtCPSF. The phenotypes of the 3' end formation esp mutants include impaired termination of the transgene transcripts, early flowering, and enhanced silencing of the FCA-beta mRNA. Based on these findings, we propose that the ESP-containing 3' end formation complexes prevent transgene and endogenous mRNAs from entering RNA-silencing pathways. According to this proposal, in the absence of these ESP proteins, these RNAs have aberrant 3' termini. The aberrant RNAs would enter the RNA silencing pathways because they are converted into dsRNA by RNA-dependent RNA polymerases.

The Drosophila master sex-switch protein Sex-lethal (SXL) regulates the splicing and/or translation of three known targets to mediate somatic sexual differentiation. Genetic studies suggest that additional target(s) of SXL exist, particularly in the female germline. Surprisingly, our detailed molecular characterization of a new potential target of SXL, enhancer of rudimentary (e(r)), reveals that SXL regulates e(r) by a novel mechanism--polyadenylation switching--specifically in the female germline. SXL binds to multiple SXL-binding sites, which include the GU-rich poly(A) enhancer, and competes for the binding of CstF64 in vitro. The SXL-binding sites are able to confer sex-specific poly(A) switching onto an otherwise nonresponsive polyadenylation signal in vivo. The sex-specific poly(A) switching of e(r) provides a means for translational regulation in germ cells. We present a model for the SXL-dependent poly(A) site choice in the female germline.

Human CstF-77 is one of the three subunits of cleavage stimulation factor (CstF) that is essential for mRNA polyadenylation. Its Drosophila homologue, suppressor of forked [su(f)], contains an intronic poly(A) site, which can lead to a short transcript without a stop codon. By both bioinformatic searches and validation with molecular biology experiments, we found that human and mouse CstF-77 genes also contain an intronic poly(A) site, which can be utilized to produce short CstF-77 transcripts lacking sequences encoding domains that are involved in many of the CstF-77 functions. The genomic sequence surrounding the poly(A) site is highly conserved among all vertebrates, but is not present in non-vertebrate species. Using public Serial Analysis of Gene Expression (SAGE) data, we found that the intronic poly(A) site is utilized in a wide range of tissues. This finding indicates that vertebrates may employ a similar alternative polyadenylation mechanism to modulate CstF-77, highlighting the importance of the regulation of CstF-77 in various species.

Polyadenylation is a process of endonucleolytic cleavage of the mRNA, followed by addition of up to 250 adenosine residues to the 3' end of the mRNA. Polyadenylation is essential for eukaryotic mRNA expression, and CstF-64 is a subunit of the CstF polyadenylation factor that is required for accurate polyadenylation. We discovered that there are two forms of the CstF-64 protein in mammalian male germ cells, one of which (CstF-64) is expressed in all tissues, the other of which (tauCstF-64) is expressed only in male germ cells and in brain (albeit at significantly lower levels in the brain). Therefore, we were surprised to find that, using reverse transcription-PCR, cDNA cloning, and RNA blot analyses, tauCstF-64 mRNA was expressed at higher levels in brain than in testis. Also, tauCstF-64 mRNA was expressed at lower but detectable levels in all tissues tested, including epididymis, heart, kidney, liver, lung, muscle, ovary, spleen, thymus, and uterus. These results suggest the hypothesis that tauCstF-64 mRNA is regulated at the translational or post-translational level.

We report here the pre-mRNA 3' end processing machinery in Entamoeba histolytica. Comparative analysis of the putative sequences participating in the pre-mRNA 3' end processing of E. histolytica genes shows similitude and differences to those described for yeast and human transcripts. By a genomic survey, we identified 16 putative genes encoding for cleavage/polyadenylation factors in this parasite. E. histolytica pre-mRNA 3' end processing machinery does not seem to contain homologous genes coding for human Symplekin, CFIm59, and CFIm68 proteins, neither sequences related to yeast Pta1p and Hrp1p. Protein sequence comparisons among E. histolytica, yeast, and human showed little variation in their functional domains through evolutive scale. E. histolytica pre-mRNA 3' end processing machinery appears to be in an intermediate evolutionary position between mammals and yeast. From these analyses, we propose a hypothetical working model for the pre-mRNA 3' end processing machinery in E. histolytica.

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.

Recent studies have uncovered new connections between the enzymes of mRNA 3' end processing and RNA polymerase II. These connections improve the efficiency of polyadenylation and signal to the polymerase to terminate transcription; their discovery reveals another level of gene regulation.

The 3' cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3' end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3' non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.