Splicing-based transcriptome-wide association studies (splicing-TWASs) of breast cancer have the potential to identify susceptibility genes. However, existing splicing-TWASs test the association of individual excised introns in breast tissue only and thus have limited power to detect susceptibility genes. In this study, we performed a multi-tissue joint splicing-TWAS that integrated splicing-TWAS signals of multiple excised introns in each gene across 11 tissues that are potentially relevant to breast cancer risk. We utilized summary statistics from a meta-analysis that combined genome-wide association study (GWAS) results of 424,650 women of European ancestry. Splicing-level prediction models were trained in GTEx (v.8) data. We identified 240 genes by the multi-tissue joint splicing-TWAS at the Bonferroni-corrected significance level; in the tissue-specific splicing-TWAS that combined TWAS signals of excised introns in genes in breast tissue only, we identified nine additional significant genes. Of these 249 genes, 88 genes in 62 loci have not been reported by previous TWASs, and 17 genes in seven loci are at least 1 Mb away from published GWAS index variants. By comparing the results of our splicing-TWASs with previous gene-expression-based TWASs that used the same summary statistics and expression prediction models trained in the same reference panel, we found that 110 genes in 70 loci that are identified only by the splicing-TWASs. Our results showed that for many genes, expression quantitative trait loci (eQTL) did not show a significant impact on breast cancer risk, whereas splicing quantitative trait loci (sQTL) showed a strong impact through intron excision events.

Splicing factors (SFs) are the major RNA-binding proteins (RBPs) and key molecules that regulate the splicing of mRNA molecules through binding to mRNAs. The expression of splicing factors is frequently deregulated in different cancer types, causing the generation of oncogenic proteins involved in cancer hallmarks. In this study, we investigated the genes that encode RNA-binding proteins and identified potential splicing factors that contribute to the aberrant splicing applying a random forest classification model. The result suggested 56 splicing factors were related to the prognosis of 13 cancers, two SF complexes in liver hepatocellular carcinoma, and one SF complex in esophageal carcinoma. Further systematic bioinformatics studies on these cancer prognostic splicing factors and their related alternative splicing events revealed the potential regulations in a cancer-specific manner. Our analysis found high ILF2-ILF3 expression correlates with poor prognosis in LIHC through alternative splicing. These findings emphasize the importance of SFs as potential indicators for prognosis or targets for therapeutic interventions. Their roles in cancer exhibit complexity and are contingent upon the specific context in which they operate. This recognition further underscores the need for a comprehensive understanding and exploration of the role of SFs in different types of cancer, paving the way for their potential utilization in prognostic assessments and the development of targeted therapies.

暂无摘要。

A large proportion of the heritability of pancreatic cancer risk remains elusive, and the contribution of specific mRNA splicing events to pancreatic cancer susceptibility has not been systematically evaluated. In this study, we performed a large splicing transcriptome-wide association study (spTWAS) using three modeling strategies (Enet, LASSO and MCP) to develop alternative splicing genetic prediction models for identifying novel susceptibility loci and splicing introns for pancreatic cancer risk by assessing 8275 pancreatic cancer cases and 6723 controls of European ancestry. Data from 305 subjects of whom the majority are of European descent in the Genotype-Tissue Expression Project (GTEx) were used and both cis-acting and promoter-enhancer interaction regions were considered to build these models. We identified nine splicing events of seven genes (ABO, UQCRC1, STARD3, ETAA1, CELA3B, LGR4 and SFT2D1) that showed an association of genetically predicted expression with pancreatic cancer risk at a false discovery rate ≤0.05. Of these genes, UQCRC1 and LGR4 have not yet been reported to be associated with pancreatic cancer risk. Fine-mapping analyses supported likely causal associations corresponding to six splicing events of three genes (P4HTM, ABO and PGAP3). Our study identified novel genes and splicing events associated with pancreatic cancer risk, which can improve our understanding of the etiology of this deadly malignancy.

Alzheimer\'s disease (AD) is a common neurodegenerative disease in aging individuals. Alternative splicing is reported to be relevant to AD development while their roles in etiology of AD remain largely elusive. We performed a comprehensive splicing transcriptome-wide association study (spTWAS) using intronic excision expression genetic prediction models of 12 brain tissues developed through three modelling strategies, to identify candidate susceptibility splicing introns for AD risk. A total of 111,326 (46,828 proxy) cases and 677,663 controls of European ancestry were studied. We identified 343 associations of 233 splicing introns (143 genes) with AD risk after Bonferroni correction (0.05/136,884 = 3.65 × 10-7). Fine-mapping analyses supported 155 likely causal associations corresponding to 83 splicing introns of 55 genes. Eighteen causal splicing introns of 15 novel genes (EIF2D, WDR33, SAP130, BYSL, EPHB6, MRPL43, VEGFB, PPP1R13B, TLN2, CLUHP3, LRRC37A4P, CRHR1, LINC02210, ZNF45-AS1, and XPNPEP3) were identified for the first time to be related to AD susceptibility. Our study identified novel genes and splicing introns associated with AD risk, which can improve our understanding of the etiology of AD.

BACKGROUND: Concurrent existence of lncRNA and circular RNA at both nucleus and cytosol within a cell at different proportions is well reported. Previous studies showed that circular RNAs are synthesized in nucleus followed by transportation across the nuclear membrane and the export is primarily defined by their length. lncRNAs primarily originated through inefficient splicing and seem to use NXF1 for cytoplasm export. However, it is not clear whether circularization of lncRNA happens only in nucleus or it also occurs in cytoplasm. Studies indicate that circular RNAs arise when the splicing apparatus undergoes a phenomenon of back splicing. Minor spliceosome (U12 type) mediated splicing occurs in cytoplasm and is responsible for the splicing of 0.5% of introns of human cells. Therefore, possibility of cRNA biogenesis mediated by minor spliceosome at cytoplasm cannot be ruled out. Secondly, information on genes transcribing both circular and lncRNAs along with total number of RBP binding sites for both of these RNA types is extractable from databases. This study showed how these apparently unconnected pieces of reports could be put together to build a model for exploring biogenesis of circular RNA.
RESULTS: As a result of this study, a model was built under the premises that, sequences with special semantics were molecular precursors in biogenesis of circular RNA which occurred through catalytic role of some specific RBPs. The model outcome was further strengthened by fulfillment of three logical lemmas which were extracted and assimilated in this work using a novel data analytic approach, Integrated Cellular Geography. Result of the study was found to be in well agreement with proposed model. Furthermore this study also indicated that biogenesis of circular RNA was a post-transcriptional event.
CONCLUSIONS: Overall, this study provides a novel systems biology based model under the paradigm of Integrated Cellular Geography which can assimilate independently performed experimental results and data published by global researchers on RNA biology to provide important information on biogenesis of circular RNAs considering lncRNAs as precursor molecule. This study also suggests the possible RBP-mediated circularization of RNA in the cytoplasm through back-splicing using minor spliceosome.

Spinal muscular atrophy (SMA) is caused by reduced levels of survival of motor neuron (SMN) protein due to deletions and/or mutations in the SMN1 gene. Risdiplam is an orally administered molecule that modifies SMN2 pre-mRNA splicing to increase functional SMN protein.
SUNFISH Part 1 was a dose-finding study conducted in 51 individuals with types 2 and 3 SMA aged 2-25 years. A dose-escalation method was used to identify the appropriate dose for the subsequent pivotal Part 2. Individuals were randomized (2:1) to risdiplam or placebo at escalating dose levels for a minimum 12-week, double-blind, placebo-controlled period, followed by treatment for 24 months. The dose selection for Part 2 was based on safety, tolerability, pharmacokinetic, and pharmacodynamic data. Exploratory efficacy was also measured.
There was no difference in safety findings for all assessed dose levels. A dose-dependent increase in blood SMN protein was observed; a median twofold increase was obtained within 4 weeks of treatment initiation at the highest dose level. The increase in SMN protein was sustained over 24 months of treatment. Exploratory efficacy showed improvement or stabilization in motor function. The pivotal dose selected for Part 2 was 5 mg for patients with a body weight ≥20 kg or 0.25 mg/kg for patients with a body weight <20 kg.
SUNFISH Part 1 demonstrated a twofold increase in SMN protein after treatment with risdiplam. The observed safety profile supported the initiation of the pivotal Part 2 study. The long-term efficacy and safety of risdiplam are being assessed with ongoing treatment.

Circular intronic RNAs (ciRNAs) are still unexplored regarding mechanisms for their emergence. We considered the ATXN2L intron lariat-derived circular RNA (ciRNA-ATXN2L) as an opportunity to conduct a cross-species examination of ciRNA genesis. To this end, we investigated 207 datasets from 4 tissues and from 13 mammalian species. While in eight species, ciRNA-ATXN2L was never detected, in pigs and rabbits, ciRNA-ATXN2L was expressed in all tissues and sometimes at very high levels. Bovine tissues were an intermediate case and in macaques and cats, only ciRNA-ATXN2L traces were detected. The pattern of ciRNA-ATXN2L restricted to only five species is not related to a particular evolution of intronic sequences. To empower our analysis, we considered 221 additional introns including 80 introns where a lariat-derived ciRNA was previously described. The primary driver of micro-ciRNA genesis (< 155 nt as ciRNA-ATXN2L) appears to be the absence of a canonical \"A\" (i.e. a \"tnA\" located in the usual branching region) to build the lariat around this adenosine. The balance between available \"non canonical-A\" (no ciRNA genesis) and \"non-A\" (ciRNA genesis) for use as a branch point to build the lariat could modify the expression level of ciRNA-ATXN2L. In addition, the rare localization of the 2\'-5\' bond in an open RNA secondary structure could also negatively affect the lifetime of ciRNAs (macaque ciRNA-ATXN2L). Our analyses suggest that ciRNA-ATXN2L is likely a functionless splice remnant. This study provides a better understanding of the ciRNAs origin, especially drivers for micro ciRNA genesis.

Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a \"putative stable association\" whose coupling might be subject to the combined regulation from both chromatin and NS changes.Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.