A Cancer-Specific Ubiquitin Ligase Drives mRNA Alternative Polyadenylation by Ubiquitinating the mRNA 3' End Processing Complex.

Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.

Involvement of oncomiRs miR-23, miR-24, and miR-27 in the regulation of alternative polyadenylation in glioblastoma via CFIm25 cleavage factor.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. The cleavage factor Im 25 (CFIm25), a crucial component of the CFIm complex, plays a key role in regulating the length of the mRNA 3'-UTR and has been implicated in various cancers, including GBM. This study sought to investigate the regulatory influence of specific microRNAs (miRNAs) on CFIm25 expression in GBM, a highly aggressive brain tumor. Bioinformatics analysis identified miRNA candidates targeting CFIm25 mRNA, and gene expression profiles from the NCBI database (GSE90603) were used for further analysis. Expression levels of CFIm25 and selected miRNAs were assessed using qRT-PCR in GBM clinical samples (n = 20) and non-malignant brain tissues (n = 5). Additionally, the MTT assay was performed to examine the effect of miRNA overexpression on U251 cell viability. Lentivectors expressing the identified miRNAs were employed to experimentally validate their regulatory role on CFIm25 in U251 cell lines, and Western blot analysis was conducted to determine CFIm25 protein levels. We observed significantly increased levels of miR-23, miR-24, and miR-27 expression, associated with a marked reduction in CFIm25 expression in GBM samples compared to non-malignant brain tissues. In particular, overexpression of miR-23, miR-24, and miR-27 in U251 cells resulted in CFIm25 downregulation at both the mRNA and protein levels, while their inhibition increased CFIm25 and reduced cell proliferation. These observations strongly implicate miR-23, miR-24, and miR-27 in regulating CFIm25 expression in GBM, emphasizing their potential as promising therapeutic targets for enhancing treatment responses in glioblastoma.

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation.

It has recently been shown that CFIm25, a canonical mRNA 3' processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3' processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25's modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3' processing factor.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

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

Mammalian cleavage factor 25 targets KLF14 to inhibit hepatic stellate cell activation and liver fibrosis.

Liver fibrosis is primarily caused by the activation of hepatic stellate cells (HSCs), which results from chronic liver damage. Understanding the pathogenesis of HSC activation could identify new therapeutic targets to treat liver fibrosis. In this study, we examined the protective role of the mammalian cleavage factor I 25 kD subunit (CFIm25, NUDT21) in inhibiting hepatic stellate cell activation. CFIm25 expression was measured in liver cirrhosis patients and a CCl4-induced mouse model. Adeno-associated viruses and adenoviruses were used to alter hepatic CFIm25 expression in vivo and in vitro to investigate how CFIm25 functions in liver fibrosis. The underlying mechanisms were explored using RNA-seq and co-IP assays. Here, we found that CFIm25 expression was drastically decreased in activated murine HSCs and fibrotic liver tissues. CFIm25 overexpression downregulated the expression of genes involved in liver fibrosis, inhibiting the progression of HSC activation, migration and proliferation. These effects resulted from direct activation of the KLF14/PPARγ signaling axis. KLF14 inhibition abrogated the CFIm25 overexpression-mediated reduction in antifibrotic effects. These data reveal that hepatic CFIm25 regulates HSC activation through the KLF14/PPARγ pathway as liver fibrosis progresses. CFIm25 may be a novel therapeutic target for liver fibrosis.

The emerging roles of CFIm25 (NUDT21/CPSF5) in human biology and disease.

The mammalian cleavage factor I subunit CFIm25 (NUDT21) binds to the UGUA sequences of precursor RNAs. Traditionally, CFIm25 is known to facilitate 3' end formation of pre-mRNAs resulting in the formation of polyadenylated transcripts. Recent studies suggest that CFIm25 may be involved in the cyclization and hence generation of circular RNAs (circRNAs) that contain UGUA motifs. These circRNAs act as competing endogenous RNAs (ceRNAs) that disrupt the ceRNA-miRNA-mRNA axis. Other emerging roles of CFIm25 include regulating both alternative splicing and alternative polyadenylation (APA). APA generates different sized transcripts that may code for different proteins, or more commonly transcripts that code for the same protein but differ in the length and sequence content of their 3' UTRs (3' UTR-APA). CFIm25 mediated global changes in 3' UTR-APA affect human physiology including spermatogenesis and the determination of cell fate. Deregulation of CFIm25 and changes in 3' UTR-APA have been implicated in several human diseases including cancer. In many cancers, CFIm25 acts as a tumor suppressor. However, there are some cancers where CFIm25 has the opposite effect. Alterations in CFIm25-driven 3' UTR-APA may also play a role in neural dysfunction and fibrosis. CFIm25 mediated 3' UTR-APA changes can be used to generate specific signatures that can be used as potential biomarkers in development and disease. Due to the emerging role of CFIm25 as a regulator of the aforementioned RNA processing events, modulation of CFIm25 levels may be a novel viable therapeutic approach. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease.

Unraveling the relevance of the polyadenylation factor EhCFIm25 in Entamoeba histolytica through proteomic analysis.

We recently reported that silencing of the polyadenylation factor EhCFIm25 in Entamoeba histolytica, the protozoan which causes human amoebiasis, affects trophozoite proliferation, death, and virulence, suggesting that EhCFIm25 may have potential as a new biochemical target. Here, we performed a shotgun proteomic analysis to identify modulated proteins that could explain this phenotype. Data are available via ProteomeXchange with identifier PXD027784. Our results revealed changes in the abundance of 75 proteins. Interestingly, STRING analysis, functional GO-term annotations, KEGG analyses, and literature review showed that modulated proteins are mainly related to glycolysis and carbon metabolism, cytoskeleton dynamics, and parasite virulence, as well as gene expression and protein modifications. Further studies are needed to confirm the hypotheses emerging from this proteomic analysis, to thereby acquire a comprehensive view of the molecular mechanisms involved.

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.

Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation.

We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (Gennarino et al., 2015). However, the patients' CNVs also included other genes. To determine if reduced NUDT21 function alone can cause disease, we generated Nudt21+/- mice to mimic NUDT21-deletion patients. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits, cortical hyperexcitability, and misregulated alternative polyadenylation (APA) in their hippocampi. Further, to determine the mediators driving neural dysfunction in humans, we partially inhibited NUDT21 in human stem cell-derived neurons to reduce CFIm25 by 30%. This induced APA and protein level misregulation in hundreds of genes, a number of which cause intellectual disability when mutated. Altogether, these results show that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.

CFIm25 and alternative polyadenylation: Conflicting roles in cancer.

Alternative polyadenylation (APA) is now widely recognized to regulate gene expression. APA is an RNA-processing mechanism that generates distinct 3' termini on mRNAs, producing mRNA isoforms. Different factors influence the initiation and development of this process. CFIm25 (among others) is a cleavage and polyadenylation factor that plays a key role in the regulation of APA. Shortening of the 3'UTRs on mRNAs leads to enhanced cellular proliferation and tumorigenicity. One reason may be the up-regulation of growth promoting factors, such as Cyclin D1. Different studies have reported a dual role of CFIm25 in cancer (both oncogenic and tumor suppressor). microRNAs (miRNAs) may be involved in CFIm25 function as well as competing endogenous RNAs (ceRNAs). The present review focuses on the role of CFIm25 in cancer, cancer treatment, and possible involvement in other human diseases. We highlight the involvement of miRNAs and ceRNAs in the function of CFIm25 to affect gene expression. The lack of understanding of the mechanisms and regulation of CFIm25 and APA has underscored the need for further research regarding their role in cancer and other diseases.

CFIm25 inhibits hepatocellular carcinoma metastasis by suppressing the p38 and JNK/c-Jun signaling pathways.

Alternative polyadenylation (APA), a post-transcriptional modification, has been implicated in many diseases, but especially in tumor proliferation. CFIm25, the 25 kDa subunit of human cleavage factor Im (CFIm), is a key factor in APA. We show that CFIm25 expression is reduced in human hepatocellular carcinoma (HCC), and its expression correlates with metastasis. Kaplan-Meier analysis indicated that CFIm25 is related to overall survival in HCC. Moreover, CFIm25 expression is negatively related to the metastatic potential of HCC cell lines. CFIm25 knockdown promotes cell invasion and migration in vitro, while overexpression of CFIm25 inhibits cell invasion and migration in vitro and inhibits intrahepatic and lung metastasis in vivo. Additional studies showed that CFIm25 disrupts epithelial-mesenchymal transition by increasing E-cadherin, that it inhibits HCC cell migration and invasion by blocking the p38 and JNK/c-Jun signaling pathways, and that CFIm25 knockdown increases the transcriptional activity of activating protein-1 (AP-1). These findings indicate that therapy directed at increasing CFIm25 expression is a potential HCC treatment.

Effect of CFIm25 knockout on RNA polymerase II transcription.

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.

Implications of polyadenylation in health and disease.

Polyadenylation is the RNA processing step that completes the maturation of nearly all eukaryotic mRNAs. It is a two-step nuclear process that involves an endonucleolytic cleavage of the pre-mRNA at the 3'-end and the polymerization of a polyadenosine (polyA) tail, which is fundamental for mRNA stability, nuclear export and efficient translation during development. The core molecular machinery responsible for the definition of a polyA site includes several recognition, cleavage and polyadenylation factors that identify and act on a given polyA signal present in a pre-mRNA, usually an AAUAAA hexamer or similar sequence. This mechanism is tightly regulated by other cis-acting elements and trans-acting factors, and its misregulation can cause inefficient gene expression and may ultimately lead to disease. The majority of genes generate multiple mRNAs as a result of alternative polyadenylation in the 3'-untranslated region. The variable lengths of the 3' untranslated regions created by alternative polyadenylation are a recognizable target for differential regulation and clearly affect the fate of the transcript, ultimately modulating the expression of the gene. Over the past few years, several studies have highlighted the importance of polyadenylation and alternative polyadenylation in gene expression and their impact in a variety of physiological conditions, as well as in several illnesses. Abnormalities in the 3'-end processing mechanisms thus represent a common feature among many oncological, immunological, neurological and hematological disorders, but slight imbalances can lead to the natural establishment of a specific cellular state. This review addresses the key steps of polyadenylation and alternative polyadenylation in different cellular conditions and diseases focusing on the molecular effectors that ensure a faultless pre-mRNA 3' end formation.