CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1.

Platinum-based chemotherapy is the standard postoperative adjuvant treatment for ovarian cancer (OC). Despite the initial response to chemotherapy, 85% of advanced OC patients will have recurrent disease. Relapsed disease and platinum resistance are the major causes of death in OC patients. In this study, we compared the global regulation of alternative polyadenylation (APA) in platinum-resistant and platinum-sensitive tissues of OC patients by analyzing a set of single-cell RNA sequencing (scRNA-seq) data from public databases and found that platinum-resistant patients exhibited global 3' untranslated region (UTR) shortening due to the different usage of polyadenylation sites (PASs). The APA regulator CSTF3 was the most significantly upregulated gene in epithelial cells of platinum-resistant OC. CSTF3 knockdown increased the sensitivity of OC cells to platinum. The lncRNA NEAT1 has two isoforms, short (NEAT1_1) and long (NEAT1_2) transcript, because of the APA processing in 3'UTR. We found that CSTF3 knockdown reduced the usage of NEAT1 proximal PAS to lengthen the transcript and facilitate the expression of NEAT1_2. Downregulation of the expression of NEAT1 (NEAT1_1/_2), but not only NEAT1_2, also increased the sensitivity of OC cells to platinum. Overexpressed NEAT1_1 reversed the platinum resistance of OC cells after knocking down CSTF3 expression. Furthermore, downregulated expression of CSTF3 and NEAT1_1, rather than NEAT1_2, was positively correlated with inactivation of the PI3K/AKT/mTOR pathway in OC cells. Together, our findings revealed a novel mechanism of APA regulation in platinum-resistant OC. CSTF3 directly bound downstream of the NEAT1 proximal PAS to generate the short isoform NEAT1_1 and was conducive to platinum resistance, which provides a potential biomarker and therapeutic strategy for platinum-resistant OC patients.

CPSF3 regulates alternative polyadenylation of CNIH2 to promote esophageal squamous cell carcinoma progression.

Alternative polyadenylation (APA), an important post-transcriptional regulatory mechanism, is aberrantly activated in cancer，but how APA functions in tumorigenesis remains elusive. We analyzed APA events in RNA-seq data in TCGA and reported 3'UTR alterations associated with esophageal squamous cell carcinoma (ESCC) patient prognosis and gene expression changes involving loss of tumor-suppressive miRNA binding sites. Moreover, we investigated the expression and function of cleavage and polyadenylation specific factor 3 (CPSF3), a key APA regulator in ESCC. By immunohistochemistry and qRT-PCR, we found that CPSF3 was highly expressed in ESCC tissues and associated with poor patient prognosis. Overexpression of CPSF3 enhanced, while knockdown of CPSF3 inhibited ESCC cell proliferation and migration in vitro and in vivo, as determined by colony formation, transwell assays and animal experiments. Iso-Seq and RNA-seq data analysis indicated that knockdown of CPSF3 favored use of the distal poly (A) site in the 3'UTR of Cornichon family AMPA receptor auxiliary protein 2 (CNIH2), resulting in a long-3'UTR CNIH2 isoform that produced less CNIH2 protein due to miR-125a-5p targeting and downregulating CNIH2 mRNA through a miR-125a-5p binding site in the long CNIH2 mRNA 3'UTR. Moreover, CPSF3-induced ESCC tumorigenicity was mediated by CNIH2. Taken together, CPSF3 promotes ESCC progression by upregulating CNIH2 expression through loss of miR-125a-5p-mediated CNIH2 repression through alternative splicing and polyadenylation of the CNIH2 mRNA 3'UTR.

Cleavage and polyadenylation machinery as a novel targetable vulnerability for human cancer.

The role of alternative polyadenylation of mRNA in sustaining aggressive features of tumors is quite well established, as it is responsible for the 3'UTR shortening of oncogenes and subsequent relief from miRNA-mediated repression observed in cancer cells. However, the information regarding the vulnerability of cancer cells to the inhibition of cleavage and polyadenylation (CPA) machinery is very scattered. Only few recent reports show the antitumor activity of pharmacological inhibitors of CPSF3, one among CPA factors. More in general, the fact that deregulated CPA can be seen as a new hallmark of cancer and as a potential reservoir of novel therapeutic targets has never been formalized. Here, to extend our view on the potential of CPA inhibition (CPAi) approaches as anticancer therapies, we systematically tested the fitness of about one thousand cell lines of different cancer types upon depletion of all known CPA factors by interrogating genome-scale CRISPR and RNAi dependency maps of the DepMap project. Our analysis confirmed core and accessory CPA factors as novel vulnerabilities for human cancer, thus highlighting the potential of CPAi as anticancer therapy. Among all, CPSF1 appeared as a promising actionable candidate for drug development, as it showed low dependency scores pancancer and particularly in highly proliferating cells. In a personalized medicine perspective, the observed differential vulnerability of cancer cell lines to selected CPA factors may be used to build up signatures to predict response of individual human tumors to CPAi approaches.

Molecular basis of human poly(A) polymerase recruitment by mPSF.

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

Predictive Value of CFIm25 Expression in Peripheral Blood Monocytes for Coronary Atherosclerosis.

Background: Cleavage factor Im25 (CFIm25) regulates cell function by affecting mRNA editing processes and plays diverse roles in various diseases. Studies have found that peripheral blood monocytes are valuable in diagnosing and prognosing coronary atherosclerosis. However, no studies have examined the predictive value of CFIm25 expression in peripheral blood monocytes for coronary atherosclerosis. Methods and Results: We collected the coronary angiography results of 267 patients and calculated the Gensini score to evaluate their degree of coronary atherosclerosis. We isolated peripheral blood monocytes and detected CFIm25 RNA expression. Based on their Gensini score, we divided the patients into negative (0, n = 46), mild lesion (≤ 8, n = 71), moderate lesion (8-23, n = 76), and severe lesion (≥ 23, n = 74) groups. Results showed that CFIm25 expression correlated negatively with the Gensini score and the number of involved coronary vessels. Univariate and multivariate binary logistic regression analyses showed that CFIm25 expression in peripheral blood monocytes was a protective factor for severe lesions, ≥ 50% stenosis, and three-vessel lesions. The areas under the receiver operating characteristic curve of CFIm25 expression for predicting lesions, severe lesions, ≥50% stenosis, and three-vessel lesions were 0.743, 0.735, 0.791, and 0.736, respectively. Conclusions: CFIm25 expression in peripheral blood monocytes correlates negatively with the degree of coronary atherosclerosis and helps predict the severity and number of coronary artery lesions.

NUDT21 interacts with NDUFS2 to activate the PI3K/AKT pathway and promotes pancreatic cancer pathogenesis.

BACKGROUND: NUDT21 (Nudix Hydrolase 21) has been shown to play an essential role in multiple biological processes. Pancreatic adenocarcinoma (PAAD) is one of the most fatal cancers in the world. However, the biological function of NUDT21 in PAAD remains rarely understood. The aim of this research was to identify the prediction value of NUDT21 in diagnosis, prognosis, immune infiltration, and signal pathway in PAAD. METHODS: Combined with the data in online databases, we analyzed the expression, immune infiltration, function enrichment, signal pathway, diagnosis, and prognosis of NUDT21 in PAAD. Then, the biological function of NUDT21 and its interacted protein in PAAD was identified through plasmid transduction system and protein mass spectrometry. Expression of NUDT21 was further verified in clinical specimens by immunofluorescence. RESULTS: We found that NUDT21 was upregulated in PAAD tissues and was significantly associated with the diagnosis and prognosis of pancreatic cancer through bioinformatic data analysis. We also found that overexpression of NUDT21 enhanced PAAD cells proliferation and migration, whereas knockdown NUDT21 restored the effects through in vitro experiment. Moreover, NDUFS2 was recognized as a potential target of NUDT21.We further verified that the expression of NDUFS2 was positively correlated with NUDT21 in PAAD clinical specimens. Mechanically, we found that NUDT21 stabilizes NDUFS2 and activates the PI3K-AKT signaling pathway. CONCLUSION: Our investigation reveals that NUDT21 is a previously unrecognized oncogenic factor in the diagnosis, prognosis, and treatment target of PAAD, and we suggest that NUDT21 might be a novel therapeutic target in PAAD.

Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3.

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.

LINC00921 reduces lung cancer radiosensitivity by destabilizing NUDT21 and driving aberrant MED23 alternative polyadenylation.

Alternative polyadenylation (APA) plays a major role in controlling transcriptome diversity and therapeutic resistance of cancers. However, long non-coding RNAs (lncRNAs) involved in pathological APA remain poorly defined. Here, we functionally characterize LINC00921, a MED13L/P300-induced oncogenic lncRNA, and show that it is required for global regulation of APA in non-small cell lung cancer (NSCLC). LINC00921 shows significant potential for reducing NSCLC radiosensitivity, and high LINC00921 levels are associated with a poor prognosis for patients with NSCLC treated with radiotherapy. LINC00921 controls NUDT21 stability by facilitating binding of NUDT21 with the E3 ligase TRIP12. LINC00921-induced destabilization of NUDT21 promotes 3' UTR shortening of MED23 mRNA via APA, which, in turn, leads to elevated MED23 protein levels in cancer cells and nuclear translocation of ß-catenin and thereby activates expression of multiple ß-catenin/T cell factor (TCF)/lymphoid enhancer-binding factor (LEF)-regulated core oncogenes (c-Myc, CCND1, and BMP4). These findings highlight the importance of functionally annotating lncRNAs controlling APA and suggest the clinical potential of therapeutics for advanced NSCLC.

Therapeutic targeting of CPSF3-dependent transcriptional termination in ovarian cancer.

Transcriptional dysregulation is a recurring pathogenic hallmark and an emerging therapeutic vulnerability in ovarian cancer. Here, we demonstrated that ovarian cancer exhibited a unique dependency on the regulatory machinery of transcriptional termination, particularly, cleavage and polyadenylation specificity factor (CPSF) complex. Genetic abrogation of multiple CPSF subunits substantially hampered neoplastic cell viability, and we presented evidence that their indispensable roles converged on the endonuclease CPSF3. Mechanistically, CPSF perturbation resulted in lengthened 3'-untranslated regions, diminished intronic polyadenylation and widespread transcriptional readthrough, and consequently suppressed oncogenic pathways. Furthermore, we reported the development of specific CPSF3 inhibitors building upon the benzoxaborole scaffold, which exerted potent antitumor activity. Notably, CPSF3 blockade effectively exacerbated genomic instability by down-regulating DNA damage repair genes and thus acted in synergy with poly(adenosine 5'-diphosphate-ribose) polymerase inhibition. These findings establish CPSF3-dependent transcriptional termination as an exploitable driving mechanism of ovarian cancer and provide a promising class of boron-containing compounds for targeting transcription-addicted human malignancies.

Molecular details of the CPSF73-CPSF100 C-terminal heterodimer and interaction with Symplekin.

Eukaryotic pre-mRNA is processed by a large multiprotein complex to accurately cleave the 3' end, and to catalyse the addition of the poly(A) tail. Within this cleavage and polyadenylation specificity factor (CPSF) machinery, the CPSF73/CPSF3 endonuclease subunit directly contacts both CPSF100/CPSF2 and the scaffold protein Symplekin to form a subcomplex known as the core cleavage complex or mammalian cleavage factor. Here we have taken advantage of a stable CPSF73-CPSF100 minimal heterodimer from Encephalitozoon cuniculi to determine the solution structure formed by the first and second C-terminal domain (CTD1 and CTD2) of both proteins. We find a large number of contacts between both proteins in the complex, and notably in the region between CTD1 and CTD2. A similarity is also observed between CTD2 and the TATA-box binding protein (TBP) domains. Separately, we have determined the structure of the terminal CTD3 domain of CPSF73, which also belongs to the TBP domain family and is connected by a flexible linker to the rest of CPSF73. Biochemical assays demonstrate a key role for the CTD3 of CPSF73 in binding Symplekin, and structural models of the trimeric complex from other species allow for comparative analysis and support an overall conserved architecture.

Alternative polyadenylation reprogramming of MORC2 induced by NUDT21 loss promotes KIRC carcinogenesis.

Alternative polyadenylation (APA), a posttranscriptional mechanism of gene expression via determination of 3'UTR length, has an emerging role in carcinogenesis. Although abundant APA reprogramming is found in kidney renal clear cell carcinoma (KIRC), which is one of the major malignancies, whether APA functions in KIRC remains unknown. Herein, we found that chromatin modifier MORC2 gained oncogenic potential in KIRC among the genes with APA reprogramming, and moreover, its oncogenic potential was enhanced by 3'UTR shortening through stabilization of MORC2 mRNA. MORC2 was found to function in KIRC by downregulating tumor suppressor DAPK1 via DNA methylation. Mechanistically, MORC2 recruited DNMT3A to facilitate hypermethylation of the DAPK1 promoter, which was strengthened by 3'UTR shortening of MORC2. Furthermore, loss of APA regulator NUDT21, which was induced by DNMT3B-mediated promoter methylation, was identified as responsible for 3'UTR shortening of MORC2 in KIRC. Additionally, NUDT21 was confirmed to act as a tumor suppressor mainly depending on downregulation of MORC2. Finally, we designed an antisense oligonucleotide (ASO) to enhance NUDT21 expression and validated its antitumor effect in vivo and in vitro. This study uncovers the DNMT3B/NUDT21/APA/MORC2/DAPK1 regulatory axis in KIRC, disclosing the role of APA in KIRC and the crosstalk between DNA methylation and APA.

NUDT21 alters glioma migration through differential alternative polyadenylation of LAMC1.

PURPOSE: Gliomas and their surrounding microenvironment constantly interact to promote tumorigenicity, yet the underlying posttranscriptional regulatory mechanisms that govern this interplay are poorly understood. METHODS: Utilizing our established PAC-seq approach and PolyAMiner bioinformatic analysis pipeline, we deciphered the NUDT21-mediated differential APA dynamics in glioma cells. RESULTS: We identified LAMC1 as a critical NUDT21 alternative polyadenylation (APA) target, common in several core glioma-driving signaling pathways. qRT-PCR analysis confirmed that NUDT21-knockdown in glioma cells results in the preferred usage of the proximal polyA signal (PAS) of LAMC1. Functional studies revealed that NUDT21-knockdown-induced 3'UTR shortening of LAMC1 is sufficient to cause translational gain, as LAMC1 protein is upregulated in these cells compared to their respective controls. We demonstrate that 3'UTR shortening of LAMC1 after NUDT21 knockdown removes binding sites for miR-124/506, thereby relieving potent miRNA-based repression of LAMC1 expression. Remarkably, we report that the knockdown of NUDT21 significantly promoted glioma cell migration and that co-depletion of LAMC1 with NUDT21 abolished this effect. Lastly, we observed that LAMC1 3'UTR shortening predicts poor prognosis of low-grade glioma patients from The Cancer Genome Atlas. CONCLUSION: This study identifies NUDT21 as a core alternative polyadenylation factor that regulates the tumor microenvironment through differential APA and loss of miR-124/506 inhibition of LAMC1. Knockdown of NUDT21 in GBM cells mediates 3'UTR shortening of LAMC1, contributing to an increase in LAMC1, increased glioma cell migration/invasion, and a poor prognosis.

Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE and POLYADENYLATION SPECIFICITY FACTOR 73.

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. The study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on an AN3661-containing medium was lethal; however, 7-d-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signal recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' untranslated region lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets. Overall, this work demonstrates that AtCPSF73 plays important part in co-transcriptional regulation, affecting growth, and development in Arabidopsis.

Synergistic inhibition of NUDT21 by secretory S100A11 and exosomal miR-487a-5p promotes melanoma oligo- to poly-metastatic progression.

Although early diagnosis and therapeutic advances have transformed the living quality and outcome of cancer patients, the poor prognosis for metastatic patients has not been significantly improved. Mechanisms underlying the complexity of metastasis cannot be simply determined by the straightforward 'cause-and-effect relationships'. We have developed a 'dry-lab-driven knowledge discovery and wet-lab validation' approach to embrace the complexity of cancer and metastasis. We have revealed for the first time that polymetastatic (POL) melanoma cells can utilize both the secretory protein pathway (S100A11-Sec23a) and the exosomal crosstalk (miR-487a-5p) to transfer their 'polymetastatic competency' to the oligometastatic (OL) melanoma cells, via synergistic co-targeting of the tumor-suppressor Nudt21. The downstream deregulated glycolysis was verified to regulate metastatic colonization efficiency. Further, two gene sets conferring independent prognosis in melanoma were identified, which have the potential for clinical translation and merit future clinical validation.

The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs.

Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Expression patterns of eight RNA-modified regulators correlating with immune infiltrates during the progression of osteoarthritis.

Background: RNA modifications in eukaryotic cells have emerged as an exciting but under-explored area in recent years and are considered to be associated with many human diseases. While several studies have been published relating to m6A in osteoarthritis (OA), we only have limited knowledge of other kinds of RNA modifications. Our study investigated eight RNA modifiers' specific roles in OA including A-to-I, APA, m5C, m6A, m7G, mcm5s2U, Nm and Ψ together with their relationship with immune infiltration. Methods: RNA modification patterns in OA samples were identified based on eight-type RNA modifiers and their correlation with the degree of immune infiltration was also methodically investigated. Receiver operating characteristic curves (ROC) and qRT-PCR was performed to confirm the abnormal expression of hub genes. The RNA modification score (Rmscore) was generated by the applications of principal component analysis (PCA) algorithm in order to quantify RNA modification modes in individual OA patients. Results: We identified 21 differentially-expressed RNA modification related genes between OA and healthy samples. For example, CFI, CBLL1 and ALKBH8 were expressed at high levels in OA (P<0.001), while RPUSD4, PUS1, NUDT21, FBL and WDR4 were expressed at low levels (P<0.001). Two candidate RNA modification regulators (WDR4 and CFI) were screened out utilizing a random forest machine learning model. We then identified two distinctive RNA modification modes in OA which were found to display distinctive biological features. High Rmscore, characterized by increased immune cell infiltration, indicated an inflamed phenotype. Conclusions: Our study was the first to systematically reveal the crosstalk and dysregulations eight-type of RNA modifications in OA. Assessing individuals' RNA modification patterns will be conductive to enhance our understanding of the properties of immune infiltration, provide novel diagnostic and prognostic biomarkers, and guide more effective immunotherapy strategies in the future.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-ß-lactamase domain and a ß-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

1H, 15N and 13C resonance assignments of a minimal CPSF73-CPSF100 C-terminal heterodimer.

The initial pre-mRNA transcript in eukaryotes is processed by a large multi-protein complex in order to correctly cleave the 3' end, and to subsequently add the polyadenosine tail. This cleavage and polyadenylation specificity factor (CPSF) is composed of separate subunits, with structural information available for both isolated subunits and also larger assembled complexes. Nevertheless, certain key components of CPSF still lack high-resolution atomic data. One such region is the heterodimer formed between the first and second C-terminal domains of the endonuclease CPSF73, with those from the catalytically inactive CPSF100. Here we report the backbone and sidechain resonance assignments of a minimal C-terminal heterodimer of CPSF73-CPSF100 derived from the parasite Encephalitozoon cuniculi. The assignment process used several amino-acid specific labeling strategies, and the chemical shift values allow for secondary structure prediction.

CFIm-mediated alternative polyadenylation safeguards the development of mammalian pre-implantation embryos.

Alternative polyadenylation (APA) gives rise to transcripts with distinct 3' untranslated regions (3' UTRs), thereby affecting the fate of mRNAs. APA is strongly associated with cell proliferation and differentiation status, and thus likely plays a critical role in the embryo development. However, the pattern of APA in mammalian early embryos is still unknown. Here, we analyzed the 3' UTR lengths in human and mouse pre-implantation embryos using available single cell RNA-seq datasets and explored the underlying mechanism driving the changes. Although human and mouse early embryos displayed distinct patterns of 3' UTR changing, RNA metabolism pathways were involved in both species. The 3' UTR lengths are likely determined by the abundance of the cleavage factor I complex (CFIm) components NUDT21 and CPSF6 in the nucleus. Importantly, depletion of either component resulted in early embryo development arrest and 3' UTR shortening. Collectively, these data highlight an essential role for APA in the development of mammalian early embryos.