-
BioRxiv : the Preprint Server For... Jun 2024Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the...
Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of , a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.
PubMed: 38915624
DOI: 10.1101/2024.06.12.598725 -
BioRxiv : the Preprint Server For... Jun 2024Genetic regulation of alternative splicing constitutes an important link between genetic variation and disease. Nonetheless, RNA splicing is regulated by both -acting...
Genetic regulation of alternative splicing constitutes an important link between genetic variation and disease. Nonetheless, RNA splicing is regulated by both -acting elements and -acting splicing factors. Determining splicing events that are directed primarily by the - or -acting mechanisms will greatly inform our understanding of the genetic basis of disease. Here, we show that long-read RNA-seq, combined with our new method isoLASER, enables a clear segregation of - and -directed splicing events for individual samples. The genetic linkage of splicing is largely individual-specific, in stark contrast to the tissue-specific pattern of splicing profiles. Analysis of long-read RNA-seq data from human and mouse revealed thousands of -directed splicing events susceptible to genetic regulation. We highlight such events in the HLA genes whose analysis was challenging with short-read data. We also highlight novel -directed splicing events in Alzheimer's disease-relevant genes such as and . Together, the clear demarcation of - and -directed splicing paves ways for future studies of the genetic basis of disease.
PubMed: 38915585
DOI: 10.1101/2024.06.14.599101 -
BioRxiv : the Preprint Server For... Jun 2024Cell type-specific alternative splicing (AS) enables differential gene isoform expression between diverse neuron types with distinct identities and functions. Current...
Cell type-specific alternative splicing (AS) enables differential gene isoform expression between diverse neuron types with distinct identities and functions. Current studies linking individual RNA-binding proteins (RBPs) to AS in a few neuron types underscore the need for holistic modeling. Here, we use network reverse engineering to derive a map of the neuron type-specific AS regulatory landscape from 133 mouse neocortical cell types defined by single-cell transcriptomes. This approach reliably inferred the regulons of 350 RBPs and their cell type-specific activities. Our analysis revealed driving factors delineating neuronal identities, among which we validated Elavl2 as a key RBP for MGE-specific splicing in GABAergic interneurons using an in vitro ESC differentiation system. We also identified a module of exons and candidate regulators specific for long- and short-projection neurons across multiple neuronal classes. This study provides a resource for elucidating splicing regulatory programs that drive neuronal molecular diversity, including those that do not align with gene expression-based classifications.
PubMed: 38915499
DOI: 10.1101/2024.06.13.597128 -
BMC Genomics Jun 2024Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However,...
BACKGROUND
Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However, the suitability and accuracy of these tools may vary when analyzing data from different species, such as humans, animals, plants, fungi, and bacteria. For most laboratory researchers lacking a background in information science, determining how to construct an analysis workflow that meets their specific needs from the array of complex analytical tools available poses a significant challenge.
RESULTS
By utilizing RNA-seq data from plants, animals, and fungi, it was observed that different analytical tools demonstrate some variations in performance when applied to different species. A comprehensive experiment was conducted specifically for analyzing plant pathogenic fungal data, focusing on differential gene analysis as the ultimate goal. In this study, 288 pipelines using different tools were applied to analyze five fungal RNA-seq datasets, and the performance of their results was evaluated based on simulation. This led to the establishment of a relatively universal and superior fungal RNA-seq analysis pipeline that can serve as a reference, and certain standards for selecting analysis tools were derived for reference. Additionally, we compared various tools for alternative splicing analysis. The results based on simulated data indicated that rMATS remained the optimal choice, although consideration could be given to supplementing with tools such as SpliceWiz.
CONCLUSION
The experimental results demonstrate that, in comparison to the default software parameter configurations, the analysis combination results after tuning can provide more accurate biological insights. It is beneficial to carefully select suitable analysis software based on the data, rather than indiscriminately choosing tools, in order to achieve high-quality analysis results more efficiently.
Topics: Workflow; RNA-Seq; Software; Fungi; Computational Biology; Sequence Analysis, RNA; Alternative Splicing
PubMed: 38914930
DOI: 10.1186/s12864-024-10414-y -
Scientific Reports Jun 2024Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for...
Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for regulating the alternative splicing of its own mRNA and ensuring that its expression is balanced to maintain homeostasis. Moreover, the exon skipping of SRSF3 leads to the production of a truncated protein instead of a frameshift mutation that generates a premature termination codon (PTC). However, the precise regulatory mechanism involved in the splicing of SRSF3 remains unclear. In this study, we first established a platform for coexpressing full-length SRSF3 (SRSF3-FL) and SRSF3-PTC and further identified a specific antibody against the SRSF3-FL and truncated SRSF3 (SRSF3-TR) proteins. Next, we found that exogenously overexpressing SRSF3-FL or SRSF3-PTC failed to reverse the effects of digoxin, caffeine, or both in combination on this molecule and its targets. Endoplasmic reticulum-related pathways, transcription factors, and chemicals such as palmitic acid and phosphate were found to be involved in the regulation of SRSF3 expression. The downregulation of SRSF3-FL by palmitic acid and phosphate was mediated via different regulatory mechanisms in HeLa cells. In summary, we provide new insights into the altered expression of the SRSF3-FL and SRSF3-TR proteins for the identification of the functions of SRSF3 in cells.
Topics: Serine-Arginine Splicing Factors; Humans; HeLa Cells; Alternative Splicing; Protein Stability; Gene Expression Regulation, Neoplastic; Neoplasms; RNA, Messenger
PubMed: 38909100
DOI: 10.1038/s41598-024-64640-1 -
International Journal of Biological... 2024Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in terms of prognosis and treatment. Recent research has identified splicing deregulation as a new...
Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in terms of prognosis and treatment. Recent research has identified splicing deregulation as a new cancer hallmark. Herein, we investigated the largely uncharacterized alternative splicing profile and the key splicing factor SF3B1 in PDAC pancreatic cells and tissues as a potential discovery source of plausible drug targets and new predictive biomarkers of clinical outcome. The research involved a transcriptome-wide analysis, comparing profiles of splicing profiles in PDAC primary cells with normal ductal cells. This revealed more than 400 significant differential splicing events in genes involved in regulation of gene expression, primarily related to mRNA splicing, and metabolism of nucleic acids. PDAC cultures were highly sensitive to the SF3B1 modulators, E7107 and Pladienolide-B, showing IC50s in the low nanomolar range. These compounds induced apoptosis, associated to induction of the MCL-1/S splice variant. and reduced cell migration, associated to RON mis-splicing. In an orthotopic mouse model, E7107 showed promising results. Furthermore, we evaluated SF3B1 expression in specimens from 87 patients and found a significant association of SF3B1 expression with progression-free and overall survival. In conclusion, SF3B1 emerges as both a potential prognostic factor and therapeutic target in PDAC, impacting cell proliferation, migration, and apoptosis. These findings warrant future studies on this new therapeutic strategy against PDAC.
Topics: Humans; RNA Splicing Factors; Pancreatic Neoplasms; Animals; Mice; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Epoxy Compounds; Prognosis; Phosphoproteins; Macrolides; Apoptosis; RNA Splicing; Alternative Splicing; Female; Cell Movement
PubMed: 38904016
DOI: 10.7150/ijbs.92671 -
Genome Biology Jun 2024The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript...
BACKGROUND
The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood.
RESULTS
We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms.
CONCLUSIONS
Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.
Topics: Animals; Nonsense Mediated mRNA Decay; Alternative Splicing; Mice; Neurons; Polypyrimidine Tract-Binding Protein; Down-Regulation; Exons; Heterogeneous-Nuclear Ribonucleoproteins; RNA, Messenger; Gene Expression Regulation, Developmental; Cell Differentiation; Neurogenesis
PubMed: 38902825
DOI: 10.1186/s13059-024-03305-8 -
Molecular Brain Jun 2024Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in...
Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in AS or by alterations to AS. Therefore, measuring AS accurately and efficiently is critical for assessing molecular phenotypes, including those associated with disease. Long-read sequencing enables more accurate quantification of differentially spliced isoform expression than short-read sequencing approaches, and third-generation platforms facilitate high-throughput experiments. To assess differences in AS across the cerebellum, cortex, hippocampus, and striatum by sex, we generated and analyzed Oxford Nanopore Technologies (ONT) long-read RNA sequencing (lrRNA-Seq) C57BL/6J mouse brain cDNA libraries. From > 85 million reads that passed quality control metrics, we calculated differential gene expression (DGE), differential transcript expression (DTE), and differential transcript usage (DTU) across brain regions and by sex. We found significant DGE, DTE, and DTU across brain regions and that the cerebellum had the most differences compared to the other three regions. Additionally, we found region-specific differential splicing between sexes, with the most sex differences in DTU in the cortex and no DTU in the hippocampus. We also report on two distinct patterns of sex DTU we observed, sex-divergent and sex-specific, that could potentially help explain sex differences in the prevalence and prognosis of various neurological and psychiatric disorders in future studies. Finally, we built a Shiny web application for researchers to explore the data further. Our study provides a resource for the community; it underscores the importance of AS in biological heterogeneity and the utility of long-read sequencing to better understand AS in the brain.
Topics: Animals; Mice, Inbred C57BL; Male; Brain; Female; Sequence Analysis, RNA; RNA, Messenger; Sex Characteristics; Alternative Splicing; RNA Isoforms; Organ Specificity; Mice; Gene Expression Profiling
PubMed: 38902764
DOI: 10.1186/s13041-024-01112-7 -
MedComm Jul 2024The DNA-dependent protein kinase (DNA-PK), catalytic subunit, also known as DNA-PKcs, is complexed with the heterodimer Ku70/Ku80 to form DNA-PK holoenzyme, which is... (Review)
Review
The DNA-dependent protein kinase (DNA-PK), catalytic subunit, also known as DNA-PKcs, is complexed with the heterodimer Ku70/Ku80 to form DNA-PK holoenzyme, which is well recognized as initiator in the nonhomologous end joining (NHEJ) repair after double strand break (DSB). During NHEJ, DNA-PKcs is essential for both DNA end processing and end joining. Besides its classical function in DSB repair, DNA-PKcs also shows multifaceted functions in various biological activities such as class switch recombination (CSR) and variable (V) diversity (D) joining (J) recombination in B/T lymphocytes development, innate immunity through cGAS-STING pathway, transcription, alternative splicing, and so on, which are dependent on its function in NHEJ or not. Moreover, DNA-PKcs deficiency has been proven to be related with human diseases such as neurological pathogenesis, cancer, immunological disorder, and so on through different mechanisms. Therefore, it is imperative to summarize the latest findings about DNA-PKcs and diseases for better targeting DNA-PKcs, which have shown efficacy in cancer treatment in preclinical models. Here, we discuss the multifaceted roles of DNA-PKcs in human diseases, meanwhile, we discuss the progresses of DNA-PKcs inhibitors and their potential in clinical trials. The most updated review about DNA-PKcs will hopefully provide insights and ideas to understand DNA-PKcs associated diseases.
PubMed: 38898995
DOI: 10.1002/mco2.613 -
Scientific Reports Jun 2024Genome analysis in cancer has focused mainly on elucidating the function and regulatory mechanisms of genes that exhibit differential expression or mutation in cancer...
Genome analysis in cancer has focused mainly on elucidating the function and regulatory mechanisms of genes that exhibit differential expression or mutation in cancer samples compared to normal samples. Recently, transcriptome analysis revealed that abnormal splicing events in cancer samples could contribute to cancer pathogenesis. Moreover, splicing variants in cancer reportedly generate diverse cancer antigens. Although abnormal splicing events are expected to be potential targets in cancer immunotherapy, the exploration of such targets and their biological significance in cancer have not been fully understood. In this study, to explore subtype-specific alternative splicing events, we conducted a comprehensive analysis of splicing events for each breast cancer subtype using large-scale splicing data derived from The Cancer Genome Atlas and found subtype-specific alternative splicing patterns. Analyses indicated that genes that produce subtype-specific alternative splicing events are potential novel targets for immunotherapy against breast cancer. The subtype-specific alternative splicing events identified in this study, which were not identified by mutation or differential expression analysis, bring new significance to previously overlooked splicing events.
Topics: Humans; Alternative Splicing; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Gene Expression Profiling; Mutation; Data Analysis
PubMed: 38898123
DOI: 10.1038/s41598-024-65035-y