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The potential impact of RNA splicing abnormalities on immune regulation in endometrial cancer
RNA splicing controls the post-transcriptional level of gene expression, allowing for the synthesis of many transcripts with various configurations and roles. Variations in RNA splicing regulatory factors, including splicing factors, signaling pathways, epigenetic modifications, and environmental factors, are typically the origin of tumor-associated splicing anomalies. Furthermore, thorough literature assessments on the intricate connection between tumor-related splicing dysregulation and tumor immunity are currently lacking. Therefore, we also thoroughly discuss putative targets associated with RNA splicing in endometrial cancer (EC) and the possible impacts of aberrant RNA splicing on the immune control of tumor cells and tumor microenvironment (TME), which contributes to enhancing the utilization of immunotherapy in the management of EC and offers an alternative viewpoint for the exploration of cancer therapies and plausible prognostic indicators.
Structural insights into spliceosome fidelity: DHX35–GPATCH1- mediated rejection of aberrant splicing substrates
The spliceosome, a highly dynamic macromolecular assembly, catalyzes the precise removal of introns from pre-mRNAs. Recent studies have provided comprehensive structural insights into the step-wise assembly, catalytic splicing and final disassembly of the spliceosome. However, the molecular details of how the spliceosome recognizes and rejects suboptimal splicing substrates remained unclear. Here, we show cryo-electron microscopy structures of spliceosomal quality control complexes from a thermophilic eukaryote, Chaetomium thermophilum. The spliceosomes, henceforth termed B*Q, are stalled at a catalytically activated state but prior to the first splicing reaction due to an aberrant 5’ splice site conformation. This state is recognized by G-patch protein GPATCH1, which is docked onto PRP8-EN and -RH domains and has recruited the cognate DHX35 helicase to its U2 snRNA substrate. In B*Q, DHX35 has dissociated the U2/branch site helix, while the disassembly helicase DHX15 is docked close to its U6 RNA 3’-end substrate. Our work thus provides mechanistic insights into the concerted action of two spliceosomal helicases in maintaining splicing fidelity by priming spliceosomes that are bound to aberrant splice substrates for disassembly.
Targeting the splicing factor SNRPB inhibits endometrial cancer progression by retaining the POLD1 intron
Dysregulated alternative splicing has been closely linked to the initiation and progression of tumors. Nevertheless, the precise molecular mechanisms through which splicing factors regulate endometrial cancer progression are still not fully understood. This study demonstrated elevated expression of the splicing factor SNRPB in endometrial cancer samples. Furthermore, our findings indicate that high SNRPB expression is correlated with poor prognosis in patients with endometrial cancer. Functionally, SNRPB inhibition hindered the proliferative and metastatic capacities of endometrial cancer cells. Mechanistically, we revealed that SNRPB knockdown decreased POLD1 expression and that POLD1 intron 22 was retained after SNRPB silencing in endometrial cancer cells, as determined via RNA sequencing data analysis. The retained intron 22 of POLD1 created a premature termination codon, leading to the absence of amino acids 941–1,107 and the loss of the site of interaction with PCNA, which is essential for POLD1 enzyme activity. In addition, POLD1 depletion decreased the increase in the malignancy of endometrial cancer cells overexpressing SNRPB. Furthermore, miR-654-5p was found to bind directly to the 3′ untranslated region of SNRPB, resulting in SNRPB expression inhibition in endometrial cancer. Antisense oligonucleotide-mediated SNRPB inhibition led to a decrease in the growth capacity of a cell-derived xenograft model and a patient with endometrial cancer-derived xenograft model. Overall, SNRPB promotes the efficient splicing of POLD1 by regulating intron retention, ultimately contributing to high POLD1 expression in endometrial cancer. The oncogenic SNRPB–POLD1 axis is an interesting therapeutic target for endometrial cancer, and antisense oligonucleotide-mediated silencing of SNRPB may constitute a promising therapeutic approach for treating patients with endometrial cancer.
The contribution of genetic determinants of blood gene expression and splicing to molecular phenotypes and health outcomes
The biological mechanisms through which most nonprotein-coding genetic variants affect disease risk are unknown. To investigate gene-regulatory mechanisms, we mapped blood gene expression and splicing quantitative trait loci (QTLs) through bulk RNA sequencing in 4,732 participants and integrated protein, metabolite and lipid data from the same individuals. We identified cis-QTLs for the expression of 17,233 genes and 29,514 splicing events (in 6,853 genes). Colocalization analyses revealed 3,430 proteomic and metabolomic traits with a shared association signal with either gene expression or splicing. We quantified the relative contribution of the genetic effects at loci with shared etiology, observing 222 molecular phenotypes significantly mediated by gene expression or splicing. We uncovered gene-regulatory mechanisms at disease loci with therapeutic implications, such as WARS1 in hypertension, IL7R in dermatitis and IFNAR2 in COVID-19. Our study provides an open-access resource on the shared genetic etiology across transcriptional phenotypes, molecular traits and health outcomes in humans (https://IntervalRNA.org.uk).
Beyond genomics: using RNA-seq from dried blood spots to unlock the clinical relevance of splicing variation in a diagnostic setting
We aimed to assess the impact of splicing variants reported in our laboratory to gain insight into their clinical relevance. A total of 108 consecutive individuals, for whom 113 splicing variants had been reported, were selected for RNA-sequencing (RNA-seq), considering the gene expression in blood. A protocol was developed to perform RNA extraction and sequencing using the same sample (dried blood spots, DBS) provided for the DNA analysis, including library preparation and bioinformatic pipeline analysis. Splicing in genes of interest was inspected using IGV, with at least three unaffected individuals as controls. From the 113 variants, we confirmed an abnormal splicing in 64 variants (57%). In 15 variants (13%), we did not observe a splicing alteration. In the remaining 34 variants, no decision could be made on the splicing effect due to insufficient sample quality (21%) or a low number of reads (9%). The most common event leading to aberrant splicing was exon skipping, identified in 31 variants (48%). Other events included cryptic donor/acceptor site usage (n = 25; 39%), intron retention (n = 4; 6%), and other complex events (n = 4; 6%). In three patients, pathologically reduced enzymatic activity (measured using the same DBS) served as additional confirmation of the abnormal splicing caused by variants in HEXA, GAA, and GLA. We implemented an RNA-seq pipeline using the same sample provided for genomic testing. This multiomic approach, as implemented in our routine diagnostic processes, clarifies the clinical relevance of most of the analyzed variants and delivers more comprehensive genetic testing.
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