Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

The splicing factor CCAR1 regulates the Fanconi anemia/BRCA pathway.

The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.

Putting a finger on histidine methylation.

Specialized enzymes add methyl groups to the nitrogens of the amino acid histidine, altering the chemical properties of its imidazole ring and, in turn, the function of the modified (poly)peptide. In this issue of Genes & Development, Shimazu and colleagues (pp. 724-742) make the remarkable discovery that CARNMT1 acts as a dual-specificity histidine methyltransferase, modifying both the small-molecule dipeptide carnosine and a set of proteins, predominantly within RNA-binding C3H zinc finger (C3H ZF) motifs. As a result, CARNMT1 modulates the activity of its protein targets to affect RNA processing and metabolism, ultimately contributing an essential function during mammalian development.

Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

Defective Autophagy Initiates Malignant Transformation.

In this issue of Molecular Cell, Park et al. (2016) elegantly demonstrate that a partial defect in autophagy supports malignant transformation as it favors the production of genotoxic reactive oxygen species by mitochondria.

Spliceosomal gene mutations in myelodysplasia: molecular links to clonal abnormalities of hematopoiesis.

Genomic analyses of the myeloid malignancies and clonal disorders of hematopoiesis that may give rise to these disorders have identified that mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are among the most common targets of somatic mutations. These spliceosomal mutations often occur in a mutually exclusive manner with one another and, in aggregate, account for the most frequent class of mutations in patients with myelodysplastic syndromes (MDSs) in particular. Although substantial progress has been made in understanding the effects of several of these mutations on splicing and splice site recognition, functional connections linking the mechanistic changes in splicing induced by these mutations to the phenotypic consequences of clonal and aberrant hematopoiesis are not yet well defined. This review describes our current understanding of the mechanistic and biological effects of spliceosomal gene mutations in MDSs as well as the regulation of splicing throughout normal hematopoiesis.

[Primary myelofibrosis with double mutation in U2AF1].

A 47-year-old woman presented with subcutaneous hemorrhage. Blood tests revealed leukoerythroblastosis, anemia, and thrombocytopenia. Bone marrow biopsy led to a diagnosis of primary myelofibrosis (aaDIPSS, DIPSS-plus: intermediate-II risk). JAK2, CALR, and MPL mutations were not detected in peripheral blood, but targeted sequencing of bone marrow specimens revealed a double mutation (Q157R, S34F) in U2AF1. Allo-PBSCT was performed using an HLA-matched related donor, and post-transplantation bone marrow examination showed complete donor chimerism on day 55. Two years after allogeneic transplantation, the patient remains relapse-free. Although U2AF1 gene abnormality is known as a poor prognostic factor in primary myelofibrosis, this patient had a favorable long-term prognosis due to prompt transplantation therapy. This case highlights the importance of detailed gene mutation analysis in patients with triple-negative MF.

Disease-related thrombocytopenia in myelofibrosis is defined by distinct genetic etiologies and is associated with unique prognostic correlates.

BACKGROUND: Thrombocytopenia in patients with myelofibrosis (MF) is prognostically detrimental and poses a therapeutic challenge. MF patients with thrombocytopenia are considered high-risk by most prognostic models and their distinct phenotype has given rise to the emerging concept of cytopenic MF. Yet, the mechanisms underlying thrombocytopenia in MF are poorly understood. METHODS: This study aimed to highlight the genetic mechanisms driving low platelet counts in treatment-naive MF patients, establish their phenotypic correlates, and assess prognostic factors specific to this group of patients. RESULTS: The authors found that most patients presenting with low platelets had a clear thrombocytopenia-specific genetic abnormality involving a U2AF1 Q157 mutation, deletion 20q, molecular complexity (three or more mutations), or high-risk karyotype. Etiologic clustering did not correlate with prognosis; however, thrombocytopenic patients were found to have unique prognostic variables including low serum albumin and mutations of SRSF2 and TP53. This led to the proposal of a prognostic model (SRSF2, albumin, TP53 score) that stratifies thrombocytopenic patients as low, intermediate, or high-risk with corresponding median survivals of 93.5, 29.5, and 7.2 months, respectively. CONCLUSIONS: This study demonstrates that thrombocytopenia in MF is driven by different genetic mechanisms and is not uniformly high-risk. As novel agents with improved hematologic safety profiles enter the treatment landscape, thoughtful, risk-adapted therapeutic decisions will be required for MF patients with thrombocytopenia. LAY SUMMARY: A significant minority of patients with myelofibrosis (MF) present with low platelets. Historically, these patients have been viewed as having "high-risk" disease, but this may not be uniformly true. Our study shows that there are various different causes for low platelets in MF, some of which represent high-risk disease whereas others do not. Additionally, our study shows that genetic mutations affecting the genes SRSF2 and TP53 are uniquely problematic in this group, as is a low serum albumin level. This study helps to risk-stratify MF patients with thrombocytopenia, thereby providing more information to guide informed and individualized treatment decisions.

U2AF1 mutation connects DNA damage to the alternative splicing of RAD51 in lung adenocarcinomas.

The recurrent mutation (S34F) in splicing factor U2AF1 is frequently observed in lung adenocarcinoma, but its function remains largely unknown. To determine the mechanistic basis and consequences of U2AF1 mutations, we established non-small cell lung carcinoma A549 cell lines with exogenous expression of wildtype (U2AF1-WT) or mutant (U2AF1-S34F). Splicing analysis revealed that U2AF1-S34F mainly caused aberrant exon usage and affected splicing of numerous DNA damage repair genes. Compared to A549 cells expressing U2AF1-WT, cells expressing U2AF1-S34F showed enhanced DNA damage and cell death in response to ATR inhibitors (ATRi). Mechanistically, U2AF1-S34F induced mis-splicing and downregulation of a key homologous recombination protein RAD51. Overexpression of RAD51 could largely rescue the defective DNA damage response in cells expressing U2AF1-S34F. Moreover, A549 cells expressing U2AF1-S34F, but not U2AF1-WT, were highly sensitive to treatment even with low dose of RAD51 inhibitor on ATRi-induced DNA damage. Our results suggest that U2AF1-S34F causes mis-splicing of DNA damage repair factors in lung cancer and sensitizes cells to RAD51 inhibition.

CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression.

Pancreatic cancer is one of the most lethal malignant tumors. However, the molecular mechanisms responsible for its progression are little known. This study aimed to understand the regulatory role of CD44V3 in pancreatic cancer. A Kaplan-Meier analysis was performed to reveal the correlation between CD44/CD44V3 expression and the prognosis of pancreatic cancer patients. CD44V3 and U2AF1 were knocked down using shRNAs. The proliferation, migration, invasion, and stemness of two pancreatic cell lines, BxPC-3 and AsPC-1, were examined. The expression of CD44V3, cancer-associated markers, and the activation of AKT signaling were detected by qRT-PCR and Western blot. Both CD44 and CD44V3 expression levels were associated with a poor prognosis in pancreatic cancer patients. Interestingly, the expression of CD44V3, instead of CD44, was greatly increased in tumor tissues. CD44V3 knockdown inhibited the proliferation, migration, invasion, and stemness of cancer cells. CD44V3 splicing was regulated by U2AF1 and downregulation of U2AF1 enhanced CD44V3 expression, which promoted pancreatic cancer progression. CD44V3 is an important cancer-promoting factor, which may serve as a potential candidate for pancreatic cancer intervention.

Down regulation of U2AF1 promotes ARV7 splicing and prostate cancer progression.

The present study aims to investigate the roles of U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) in the resistance to anti-androgen treatment in prostate cancer and its underlying mechanism. U2AF1 and androgen receptor variant 7 (ARV7) knockdown and overexpression were introduced in PC3 and DU145 cells. In addition, a bicalutamide-resistant PC3 (PC3 BR) cell line was also constructed. Cell count, MTT and soft agar colony formation assays were performed to evaluate cell proliferation. qRT-PCR was applied to determine the mRNA levels of U2AF1, ARV7 and Mitogen-Activated Protein Kinase 1 (MAPK1). Western blot was used to determine the MAPK1 protein expression. A negative correlation between ARV7 and U2AF1 in prostate tumor tissues was observed. U2AF1 downregulation was correlated with poor prognosis in prostate cancer patients. U2AF1 exhibited a negative correlation with ARV7 and its downregulation promoted prostate cancer cell proliferation and bicalutamide resistance. The regulatory effects of U2AF1 on ARV7 splicing were associated with MAPK1. U2AF1 affected prostate cancer proliferation and anti-androgen resistance by regulating ARV7 splicing.

Impaired hematopoiesis and leukemia development in mice with a conditional knock-in allele of a mutant splicing factor gene U2af1.

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-ethyl-N-nitrosourea (ENU). Overall, 3 of 16 ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss- or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzf1 However, the engineered U2af1(S34F) missense mutation reverted to WT in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures.

Somatic mutations of pre-mRNA splicing factors recur among patients with myelodysplastic syndrome (MDS) and related malignancies. Although these MDS-relevant mutations alter the splicing of a subset of transcripts, the mechanisms by which these single amino acid substitutions change gene expression remain controversial. New structures of spliceosome intermediates and associated protein complexes shed light on the molecular interactions mediated by 'hotspots' of the SF3B1 and U2AF1 pre-mRNA splicing factors. The frequently mutated SF3B1 residues contact the pre-mRNA splice site. Based on structural homology with other spliceosome subunits, and recent findings of altered RNA binding by mutant U2AF1 proteins, we suggest that affected U2AF1 residues also contact pre-mRNA. Altered pre-mRNA recognition emerges as a molecular theme among MDS-relevant mutations of pre-mRNA splicing factors.

Deoxynivalenol globally affects the selection of 3' splice sites in human cells by suppressing the splicing factors, U2AF1 and SF1.

Deoxynivalenol (DON) is one of the most abundant mycotoxins and has adverse effects on several biological processes, posing risks of protein synthesis-disrupting effects and ribotoxic response. Therefore, chronic exposure to DON would fundamentally reshape the global expression pattern. Whether DON causes toxic effects on mRNA splicing, a fundamental biological process, remains unclear. In this study, we found that administration of the relative low dosage of DON dramatically changed the alternative splicing of pre-mRNA in HepG2 cells. The overall number of transcripts with aberrant selection of 3' splice sites was significantly increased in DON-exposed HepG2 cells. This effect was further confirmed in two other human cell lines, HEK293 and Caco-2, suggesting that this DON-induced alteration in splicing patterns was universal in human cells. Among these DON-induced changes in alternative splicing, the expression levels of two related splicing factors, SF1 and U2AF1, which are essential for 3' splice site recognitions, were strongly suppressed. Overexpression of either of the two splicing factors strongly alleviated the DON-induced aberrant selection of 3' splice sites. Moreover, SF1 was required for human cell proliferation in DON exposure, and the restoration of SF1 expression partially reinstated the proliferation potential for DON-treated cells. In conclusion, our study suggests that DON, even at a low dosage, has great potential to change gene expression globally by affecting not only protein synthesis but also mRNA processing in human cells.

The significance of PA28γ and U2AF1 in oral mucosal carcinogenesis.

OBJECTIVE: Proteasome activator 28γ (PA28γ) upregulation plays a critical role in the carcinogenesis of many malignancies, including oral cancer. We aim to screen the related genes of PA28γ and investigate their function in oral mucosa carcinogenesis. MATERIALS AND METHODS: Bioinformatics analysis was performed to screen the related genes of PA28γ. Immunohistochemical analysis was carried out to validate their correlation in oral squamous cell carcinoma (OSCC) and detect their expression levels in the whole process of oral mucosa carcinogenesis. The Kaplan-Meier method was used for estimating the overall survival, and the Cox models were constructed to predict the prognosis. RESULTS: U2 small nuclear RNA auxiliary factor 1 (U2AF1) was screened out, and the correlation between U2AF1 and PA28γ was further validated in OSCC. The expression levels of PA28γ and U2AF1 were gradually increased from normal to oral potentially malignant disorders (OPMD) to OSCC tissues. Overall survival was significantly shorter in patients with high U2AF1 expression and the combined application of U2AF1 and PA28γ notably improved the accuracy of prognosis prediction. CONCLUSION: U2AF1 and PA28γ might play pivotal roles in the progression of OPMD, which may provide insights into the development of new therapeutic strategies to prevent OPMD from becoming malignant.