Components Of Splicing Machinery Research Articles

Splicing Machinery Is Impaired in Oral Squamous Cell Carcinomas and Linked to Key Pathophysiological Features.

Alternative splicing dysregulation is an emerging cancer hallmark, potentially serving as a source of novel diagnostic, prognostic, or therapeutic tools. Inhibitors of the activity of the splicing machinery can exert antitumoral effects in cancer cells. We aimed to characterize the splicing machinery (SM) components in oral squamous cell carcinoma (OSCC) and to evaluate the direct impact of the inhibition of SM-activity on OSCC-cells. The expression of 59 SM-components was assessed using a prospective case-control study of tumor and healthy samples from 37 OSCC patients, and the relationship with clinical and histopathological features was assessed. The direct effect of pladienolide-B (SM-inhibitor) on the proliferation rate of primary OSCC cell cultures was evaluated. A significant dysregulation in several SM components was found in OSCC vs. adjacent-healthy tissues [i.e., 12 out of 59 (20%)], and their expression was associated with clinical and histopathological features of less aggressiveness and overall survival. Pladienolide-B treatment significantly decreased OSCC-cell proliferation. Our data reveal a significantly altered expression of several SM-components and link it to pathophysiological features, reinforcing a potential clinical and pathophysiological relevance of the SM dysregulation in OSCC. The inhibition of SM-activity might be a therapeutic avenue in OSCC, offering a clinically relevant opportunity to be explored.

Spliceosomic dysregulation in pancreatic cancer uncovers splicing factors PRPF8 and RBMX as novel candidate actionable targets.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, characterized by late diagnosis and poor treatment response. Surgery is the only curative approach, only available to early-diagnosed patients. Current therapies have limited effects, cause severe toxicities, and minimally improve overall survival. Understanding of splicing machinery alterations in PDAC remains incomplete. Here, we comprehensively examined 59 splicing machinery components, uncovering dysregulation in pre-mRNA processing factor 8 (PRPF8) and RNA-binding motif protein X-linked (RBMX). Their downregulated expression was linked to poor prognosis and malignancy features, including tumor stage, invasion and metastasis, and associated with poorer survival and the mutation of key PDAC genes. Experimental modulation of these splicing factors in pancreatic cancer cell lines reverted their expression to non-tumor levels and resulted in decreased key tumor-related features. These results provide evidence that the splicing machinery is altered in PDAC, wherein PRPF8 and RBMX emerge as candidate actionable therapeutic targets.

Intron Retention: What that means for the biologist

Introns are non-coding segments within eukaryotic genes that are transcribed along with exons. They are subsequently removed during splicing, and their presence allows for generating diverse mRNA isoforms through the process called alternative splicing. The functional significance of introns going beyond mere "junk DNA" is questionable until recently; scientists identified that these pieces of junk DNA could hang around with the functional mRNA escaping splicing. Intron retention is a type of alternative splicing, a process that occurs during the maturation of messenger RNA (mRNA) in eukaryotic cells. In eukaryotes, genes typically consist of exons (coding regions) and introns (non-coding regions). After transcription, the precursor mRNA (pre-mRNA) undergoes splicing, where introns are removed, and exons are joined together to form the mature mRNA. Intron retention, however, deviates from this typical splicing process. Instead of being entirely removed from the pre-mRNA, the mature mRNA may retain one or more introns. This results in the inclusion of intronic sequences within the final mRNA transcript. The retained introns may contain premature stop codons or regulatory elements that can influence gene expression and protein function. Intron retention is a form of alternative splicing because it generates multiple mRNA isoforms from a single gene. This diversity in mRNA transcripts can produce different protein isoforms with distinct functions or regulatory properties. The regulation of intron retention is complex and involves various cellular factors, including splicing machinery components and RNA-binding proteins. Research on intron retention has revealed its importance in various biological processes, including development, cell growth and differentiation, and diseases like cancer. Aberrant intron retention has been associated with certain genetic disorders and cancers. Understanding the mechanisms and functional consequences of intron retention is crucial for unraveling the complexity of gene regulation and its impact on cellular processes. With an increasing output of data from high throughput assays line RNA Seq, scRNA-Seq, etc., it's high time data scientists look into these signatures in the context of disease pathology. This may well be the tip of the iceberg.

THU080 Splicing Machinery Dysregulation As A Source Of Novel Diagnostic, Prognostic And Therapeutic Targets In Craniopharyngiomas

Abstract Disclosure: A.C. Fuentes-Fayos: None. M.E. Garcia-Garcia: None. D. Cano: None. A.J. Martínez-Fuentes: None. I. Di Caro: None. M.D. Gahete: None. J.P. Castaño: None. J.P. Martínez-Barbera: None. A. Soto-Moreno: None. M.A. Galvez-Moreno: None. J.M. Jimenez-Vacas: None. R.M. Luque: None. Craniopharyngiomas (CP) are relatively benign epithelial tumors that typically arise in the sellar/suprasellar region, and are classified in adamantinomatous (ACP) and papillary (PCP). Diagnosis is usually performed when tumor development is already advanced, and serious comorbidities are present. The first-line therapy is usually surgery, but frequently the resection is not complete, causing high recurrence rates. Therefore, the identification of alternative diagnostic/prognostic/therapeutic tools to improve CP management is necessary. Recently, growing evidence indicates that defects in the splicing-process are frequent in cancer, leading to the appearance of altered spliceosome components (SCs), splicing-factors (SFs) and/or aberrant splicing-variants (SVs), which are associated with the development/progression/aggressiveness of various cancer types. Therefore, our aim was to explore the putative oncogenic role of key splicing-related factors in CP through: 1) interrogating the expression profile of key splicing machinery components in ACP (n=36) and PCP (n=4) vs. control samples [n=11; normal-pituitaries (NP)]; and 2) implementing different bioinformatic and functional approaches (RNAseq and CP primary cell-cultures). Our results revealed a substantial number of SCs and SFs drastically altered in ACP vs. NP, and also when primary vs. recurrent ACP were compared. Specifically, 4 SFs were identified as the most discriminating diagnostic/prognostic factors, being corroborated in additional human cohorts, and associated with key clinical parameters suggesting a potential oncogenic role in CP. Dysregulation of 2 of these SFs was also corroborated in RNAseq of an additional cohort of 18 ACP vs. 3 NP. In vitro overexpression of these SFs in primary ACP-derived cells revealed a critical functional role of these factors in ACP. In fact, this overexpression induced the phosphorylation of MAPK and AKT pathways and reduced the phosphorylation JAK/STAT, NF-kB and TGFB that could be involved in the CP tumorigenesis. Finally, key SVs that are associated to CP development/progression (e.g., GNAS) were identified as potential oncogenic linkers with the observed splicing-machinery dysregulation. In conclusion, spliceosome is drastically altered in CP wherein some SFs could represent attractive novel diagnostic/prognostic and therapeutic targets for this endocrine pathology. Funding: Junta de Andalucia (PEER-0048-2020, Cofunded by EU-Programa Operativo FEDER 2014-2020). Presentation: Thursday, June 15, 2023

Adipose tissue plasticity in pheochromocytoma patients suggests a role of the splicing machinery in human adipose browning

Adipose tissue from pheochromocytoma patients acquires brown fat features, making it a valuable model for studying the mechanisms that control thermogenic adipose plasticity in humans. Transcriptomic analyses revealed a massive downregulation of splicing machinery components and splicing regulatory factors in browned adipose tissue from patients, with upregulation of a few genes encoding RNA-binding proteins potentially involved in splicing regulation. These changes were also observed in cell culture models of human brown adipocyte differentiation, confirming a potential involvement of splicing in the cell-autonomous control of adipose browning. The coordinated changes in splicing are associated with a profound modification in the expression levels of splicing-driven transcript isoforms for genes involved in the specialized metabolism of brown adipocytes and those encoding master transcriptional regulators of adipose browning. Splicing control appears to be a relevant component of the coordinated gene expression changes that allow human adipose tissue to acquire a brown phenotype.

Characterization of splicing machinery components in thyroid cancer and their correlation/association with tumour behaviour and clinical features

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Splicing machinery is profoundly altered in systemic lupus erythematosus and antiphospholipid syndrome and directly linked to key clinical features

ObjectivesTo characterize the splicing machinery (SM) of leukocytes from primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome with lupus (APS + SLE) patients, and to assess its clinical involvement. MethodsMonocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS + SLE) and 50 HD were purified, and 45 selected SM components were evaluated by qPCR-microfluidic array. Relationship with clinical features and underlying regulatory mechanisms were assessed. ResultsAPS, SLE and APS + SLE leukocytes displayed significant and specific alterations in SM-components (SMC), associated with clinical features [autoimmune profiles, disease activity, lupus nephritis (LN), and CV-risk markers]. A remarkable relationship among dysregulated SMC in monocytes and the presence of LN in SLE was highlighted, revealing a novel pathological mechanism, which was further explored. Immunohistology analysis of renal biopsies highlighted the pathological role of the myeloid compartment in LN. Transcriptomic analysis of monocytes from SLE-LN(+) vs SLE-LN(−) identified 271 genes differentially expressed, mainly involved in inflammation and IFN-signaling. Levels of IFN-related genes correlated with those of SMC in SLE-LN(+). These results were validated in two external SLE-LN(+) datasets of whole-blood and kidney biopsies. In vitro, SLE-LN(+)-serum promoted a concomitant dysregulation of both, the IFN signature and several SMC, further reversed by JAKinibs treatment. Interestingly, IFNs, key inflammatory cytokines in SLE pathology, also altered SMC. Lastly, the over/down-expression of selected SMC in SLE-monocytes reduced the release of inflammatory cytokines and their adhesion capacity. ConclusionOverall, we have identified, for the first time, a specific alteration of SMC in leukocytes from APS, SLE and APS + SLE patients that would be responsible for the development of distinctive clinical profiles.

PRPF8 increases the aggressiveness of hepatocellular carcinoma by regulating FAK/AKT pathway via fibronectin 1 splicing

Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.

HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.

Spliceosomic dysregulation unveils NOVA1 as a candidate actionable therapeutic target in pancreatic neuroendocrine tumors

Dysregulation of the splicing machinery is emerging as a hallmark in cancer due to its association with multiple dysfunctions in tumor cells. Inappropriate function of this machinery can generate tumor-driving splicing variants and trigger oncogenic actions. However, its role in pancreatic neuroendocrine tumors (PanNETs) is poorly defined. In this study we aimed to characterize the expression pattern of a set of splicing machinery components in PanNETs, and their relationship with aggressiveness features. A qPCR-based array was first deployed to determine the expression levels of components of the major (n = 13) and minor spliceosome (n = 4) and associated splicing factors (n = 27), using a microfluidic technology in 20 PanNETs and non-tumoral adjacent samples. Subsequently, in vivo and in vitro models were applied to explore the pathophysiological role of NOVA1. Expression analysis revealed that a substantial proportion of splicing machinery components was altered in tumors. Notably, key splicing factors were overexpressed in PanNETs samples, wherein their levels correlated with clinical and malignancy features. Using in vivo and in vitro assays, we demonstrate that one of those altered factors, NOVA1, is tightly related to cell proliferation, alters pivotal signaling pathways and interferes with responsiveness to drug treatment in PanNETs, suggesting a role for this factor in the aggressiveness of these tumors and its suitability as therapeutic target. Altogether, our results unveil a severe alteration of the splicing machinery in PanNETs and identify the putative relevance of NOVA1 in tumor development/progression, which could provide novel avenues to develop diagnostic biomarkers and therapeutic tools for this pathology.

HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention.

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.

Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis.

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes.

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

Dietary Intervention Modulates the Expression of Splicing Machinery in Cardiovascular Patients at High Risk of Type 2 Diabetes Development: From the CORDIOPREV Study.

Type-2 diabetes mellitus (T2DM) has become a major health problem worldwide. T2DM risk can be reduced with healthy dietary interventions, but the precise molecular underpinnings behind this association are still incompletely understood. We recently discovered that the expression profile of the splicing machinery is associated with the risk of T2DM development. Thus, the aim of this work was to evaluate the influence of 3-year dietary intervention in the expression pattern of the splicing machinery components in peripheral blood mononuclear cells (PBMCs) from patients within the CORDIOPREV study. Expression of splicing machinery components was determined in PBMCs, at baseline and after 3 years of follow-up, from all patients who developed T2DM (Incident-T2DM, n = 107) and 108 randomly selected non-T2DM subjects, who were randomly enrolled in two healthy dietary patterns (Mediterranean or low-fat diets). Dietary intervention modulated the expression of key splicing machinery components (i.e., up-regulation of SPFQ/RMB45/RNU6, etc., down-regulation of RNU2/SRSF6) after three years, independently of the type of healthy diet. Some of these changes (SPFQ/RMB45/SRSF6) were associated with key clinical features and were differentially induced in Incident-T2DM patients and non-T2DM subjects. This study reveals that splicing machinery can be modulated by long-term dietary intervention, and could become a valuable tool to screen the progression of T2DM.

A supervised machine learning-based methodology for analyzing dysregulation in splicing machinery: An application in cancer diagnosis.

Deregulated splicing machinery components have shown to be associated with the development of several types of cancer and, therefore, the determination of such alterations can help the development of tumor-specific molecular targets for early prognosis and therapy. Determining such splicing components, however, is not a straightforward task mainly due to the heterogeneity of tumors, the variability across samples, and the fat-short characteristic of genomic datasets. In this work, a supervised machine learning-based methodology is proposed, allowing the determination of subsets of relevant splicing components that best discriminate samples. The methodology comprises three main phases: first, a ranking of features is determined by means of applying feature weighting algorithms that compute the importance of each splicing component; second, the best subset of features that allows the induction of an accurate classifier is determined by means of conducting an effective heuristic search; then the confidence over the induced classifier is assessed by means of explaining the individual predictions and its global behavior. At the end, an extensive experimental study was conducted on a large collection of transcript-based datasets, illustrating the utility and benefit of the proposed methodology for analyzing dysregulation in splicing machinery.

Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors.

Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology.

Mutations in Splicing Factor Genes in Myeloid Malignancies: Significance and Impact on Clinical Features.

Components of the pre-messenger RNA splicing machinery are frequently mutated in myeloid malignancies. Mutations in LUC7L2, PRPF8, SF3B1, SRSF2, U2AF1, and ZRSR2 genes occur at various frequencies ranging between 40% and 85% in different subtypes of myelodysplastic syndrome (MDS) and 5% and 10% of acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). In some instances, splicing factor (SF) mutations have provided diagnostic utility and information on clinical outcomes as exemplified by SF3B1 mutations associated with increased ring sideroblasts (RS) in MDS-RS or MDS/MPN-RS with thrombocytosis. SF3B1 mutations are associated with better survival outcomes, while SRSF2 mutations are associated with a shorter survival time and increased AML progression, and U2AF1 mutations with a lower remission rate and shorter survival time. Beside the presence of mutations, transcriptomics technologies have shown that one third of genes in AML patients are differentially expressed, leading to altered transcript stability, interruption of protein function, and improper translation compared to those of healthy individuals. The detection of SF mutations demonstrates the importance of splicing abnormalities in the hematopoiesis of MDS and AML patients given the fact that abnormal splicing regulates the function of several transcriptional factors (PU.1, RUNX1, etc.) crucial in hematopoietic function. This review provides a summary of the significance of the most frequently mutated SF genes in myeloid malignancies and an update on novel targeted therapies in experimental and clinical trial stages.

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that small deletions in SRSF2 cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. We also verified aberrant splicing of key genes USP9X, USP24 (deubiquitinating enzymes), LUC7L2 (splice factor) and EED (PRC2 component) in MDS harboring small deletions of SRSF2. Pathway analysis revealed that mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations. Our study reveals splicing signatures of each splice factor mutation and identifies shared and distinct sets of mis-spliced genes and affected biological processes in different spliceosome mutant MDS.

Dysregulation of the Splicing Machinery Is Associated to the Development of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a common obesity-associated pathology characterized by hepatic fat accumulation, which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Obesity is associated with profound changes in gene-expression patterns of the liver, which could contribute to the onset of comorbidities. As these alterations might be linked to a dysregulation of the splicing process, we aimed to determine whether the dysregulation in the expression of splicing machinery components could be associated with NAFLD. We collected 41 liver biopsies from nonalcoholic individuals with obesity, with or without hepatic steatosis, who underwent bariatric surgery. The expression pattern of splicing machinery components was determined using a microfluidic quantitative PCR-based array. An in vitro approximation to determine lipid accumulation using HepG2 cells was also implemented. The liver of patients with obesity and steatosis exhibited a severe dysregulation of certain splicing machinery components compared with patients with obesity without steatosis. Nonsupervised clustering analysis allowed the identification of three molecular phenotypes of NAFLD with a unique fingerprint of alterations in splicing machinery components, which also presented distinctive hepatic and clinical-metabolic alterations and a differential response to bariatric surgery after 1 year. In addition, in vitro silencing of certain splicing machinery components (i.e., PTBP1, RBM45, SND1) reduced fat accumulation and modulated the expression of key de novo lipogenesis enzymes, whereas conversely, fat accumulation did not alter spliceosome components expression. There is a close relationship between splicing machinery dysregulation and NAFLD development, which should be further investigated to identify alternative therapeutic targets.

Changes in Splicing Machinery Components Influence, Precede, and Early Predict the Development of Type 2 Diabetes: From the CORDIOPREV Study.

BackgroundType-2 diabetes mellitus (T2DM) is a major health problem with increasing incidence, which severely impacts cardiovascular disease. Because T2DM is associated with altered gene expression and aberrant splicing, we hypothesized that dysregulations in splicing machinery could precede, contribute to, and predict T2DM development.MethodsA cohort of patients with cardiovascular disease (CORDIOPREV study) and without T2DM at baseline (at the inclusion of the study) was used (n = 215). We determined the expression of selected splicing machinery components in fasting and 4 h-postprandial peripheral blood mononuclear cells (PBMCs, obtained at baseline) from all the patients who developed T2DM during 5-years of follow-up (n = 107 incident-T2DM cases) and 108 randomly selected non-T2DM patients (controls). Serum from incident-T2DM and control patients was used to analyze in vitro the modulation of splicing machinery expression in control PBMCs from an independent cohort of healthy subjects.FindingsExpression of key splicing machinery components (e.g. RNU2, RNU4 or RNU12) from fasting and 4 h-postprandial PBMCs of incident-T2DM patients was markedly altered compared to non-T2DM controls. Moreover, in vitro treatment of healthy individuals PBMCs with serum from incident-T2DM patients (compared to non-T2DM controls) reduced the expression of splicing machinery elements found down-regulated in incident-T2DM patients PBMCs. Finally, fasting/postprandial levels of several splicing machinery components in the PBMCs of CORDIOPREV patients were associated to higher risk of T2DM (Odds Ratio > 4) and could accurately predict (AUC > 0.85) T2DM development.InterpretationOur results reveal the existence of splicing machinery alterations that precede and predict T2DM development in patients with cardiovascular disease.FundISCIII, MINECO, CIBERObn.