Splicing Patterns Research Articles

Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response

Mitochondrial morphology and function change dynamically in response to intracellular signaling and the surrounding environment. The mitochondrial fission factor Mff, which localizes to the outer mitochondrial membrane, mediates not only mitochondrial fission by recruiting the dynamin-related GTPase Drp1 to mitochondrial fission sites but also the double-stranded RNA-induced antiviral response on mitochondria through mitochondrial antiviral signaling (MAVS). Mff is reported to be regulated by AMP-activated protein kinase (AMPK)-mediated protein phosphorylation and alternative pre-mRNA splicing; however, the relationships among RNA splicing, phosphorylation, and multiple functions of Mff have not been fully understood. Here, we showed that mouse Mff has a tissue-specific splicing pattern, and at least eight Mff splice isoforms were expressed in mouse embryonic fibroblasts (MEFs). We introduced single Mff isoforms into Mff knockout MEFs and found that insertion of exon 6 just after the phosphorylation site, by the alternative splicing, reduced its phosphorylation by AMPK and its functions in mitochondrial fission and the antiviral response. In addition, the underlying mechanism repressing these functions was independent of phosphorylation. These results indicate that multiple functions of Mff on mitochondria are regulated by AMPK-mediated phosphorylation and alternative splicing, under the control of energy metabolism and cellular differentiation.

Pathomechanisms of Monoallelic variants in TTN causing skeletal muscle disease.

Pathogenic variants in the titin gene (TTN) are known to cause a wide range of cardiac and musculoskeletal disorders, with skeletal myopathy mostly attributed to biallelic variants. We identified monoallelic truncating variants (TTNtv), splice site or internal deletions in TTN in probands with mild, progressive axial and proximal weakness, with dilated cardiomyopathy frequently developing with age. These variants segregated in an autosomal dominant pattern in 7 out of 8 studied families. We investigated the impact of these variants on mRNA, protein levels, and skeletal muscle structure and function. Results reveal that nonsense-mediated decay likely prevents accumulation of harmful truncated protein in skeletal muscle in patients with TTNtvs. Splice variants and an out-of-frame deletion induce aberrant exon skipping, while an in-frame deletion produces shortened titin with intact N- and C-termini, resulting in disrupted sarcomeric structure. All variant types were associated with genome-wide changes in splicing patterns, which represent a hallmark of disease progression. Lastly, RNA-seq studies revealed that GDF11, a member of the TGF-β superfamily, is upregulated in diseased tissue, indicating that it might be a useful therapeutic target in skeletal muscle titinopathies.

A novel intron variant in the prolactin gene associated with eggshell weight and thickness with putative alternative splicing patterns in chickens

A novel intron variant in the prolactin gene associated with eggshell weight and thickness with putative alternative splicing patterns in chickens

Non-coding cause of congenital heart defects: Abnormal RNA splicing with multiple isoforms as a mechanism for heterotaxy

Heterotaxy is a disorder characterized by severe congenital heart defects (CHDs) and abnormal left-right patterning in other thoracic or abdominal organs. Clinical and research-based genetic testing has previously focused on evaluation of coding variants to identify causes of CHDs, leaving non-coding causes of CHDs largely unknown. Variants in the transcription factor zinc finger of the cerebellum 3 (ZIC3) cause X-linked heterotaxy. We identified an X-linked heterotaxy pedigree without a coding variant in ZIC3. Whole-genome sequencing revealed a deep intronic variant (ZIC3 c.1224+3286A>G) predicted to alter RNA splicing. An invitro minigene splicing assay confirmed the variant acts as a cryptic splice acceptor. CRISPR-Cas9 served to introduce the ZIC3 c.1224+3286A>G variant into human embryonic stem cells demonstrating pseudoexon inclusion caused by the variant. Surprisingly, Sanger sequencing of the resulting ZIC3 c.1224+3286A>G amplicons revealed several isoforms, many of which bypass the normal coding sequence of the third exon of ZIC3, causing a disruption of a DNA-binding domain and a nuclear localization signal. Short- and long-read mRNA sequencing confirmed these initial results and identified additional splicing patterns. Assessment of four isoforms determined abnormal functions invitro and invivo while treatment with a splice-blocking morpholino partially rescued ZIC3. These results demonstrate that pseudoexon inclusion in ZIC3 can cause heterotaxy and provide functional validation of non-coding disease causation. Our results suggest the importance of non-coding variants in heterotaxy and the need for improved methods to identify and classify non-coding variation that may contribute to CHDs.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

RNA Splicing Events in Circulation Distinguish Individuals with and without New-Onset Type 1 Diabetes.

Alterations in RNA splicing may influence protein isoform diversity that contributes to or reflects the pathophysiology of certain diseases. Whereas specific RNA splicing events in pancreatic islets have been investigated in models of inflammation in vitro, how RNA splicing in the circulation correlates with or is reflective of T1D disease pathophysiology in humans remains unexplored. To use machine learning to investigate if alternative RNA splicing events differ between individuals with and without new-onset type 1 diabetes (T1D) and to determine if these splicing events provide insight into T1D pathophysiology. RNA deep sequencing was performed on whole blood samples from two independent cohorts: a training cohort consisting of 12 individuals with new-onset T1D and 12 age- and sex-matched nondiabetic controls and a validation cohort of the same size and demographics. Machine learning analysis was used to identify specific isoforms that could distinguish individuals with T1D from controls. Distinct patterns of RNA splicing differentiated participants with T1D from unaffected controls. Notably, certain splicing events, particularly involving retained introns, showed significant association with T1D. Machine learning analysis using these splicing events as features from the training cohort demonstrated high accuracy in distinguishing between T1D subjects and controls in the validation cohort. Gene Ontology pathway enrichment analysis of the retained intron category showed evidence for a systemic viral response in T1D subjects. Alternative RNA splicing events in whole blood are significantly enriched in individuals with new-onset T1D and can effectively distinguish these individuals from unaffected controls. Our findings also suggest that RNA splicing profiles offer the potential to provide insights into disease pathogenesis.

RBPMS promotes contractile phenotype splicing in human embryonic stem cell derived vascular smooth muscle cells.

Differentiated Vascular Smooth Muscle Cells (VSMCs) express a unique network of mRNA isoforms via smooth muscle specific alternative splicing (SM-AS) in functionally critical genes, including those comprising the contractile machinery. We previously described RNA Binding Protein Multiple Splicing (RBPMS) as a potent driver of differentiated SM-AS in the rat PAC1 VSMC cell line. What is unknown is how RBPMS affects VSMC phenotype and behaviour. Here, we aimed to dissect the role of RBPMS in SM-AS in human cells and determine the impact on VSMC phenotypic properties. We used human embryonic stem cell-derived VSMCs (hESC-VSMCs) as our platform. hESC-VSMCs are inherently immature and we found that they display only partially differentiated SM-AS patterns while RBPMS protein levels are low. We found that RBPMS overexpression induces SM-AS patterns in hESC-VSMCs akin to the contractile tissue VSMC splicing patterns. We present in silico and experimental findings that support RBPMS' splicing activity as mediated through direct binding and via functional cooperativity with splicing factor RBFOX2 on a significant subset of targets. We also demonstrate that RBPMS can alter the motility and the proliferative properties of hESC-VSMCs to mimic a more differentiated state. Overall, this study emphasizes a critical role for RBPMS in establishing the contractile phenotype splicing program of human VSMCs.

Pangenome Identification and Analysis of Terpene Synthase Gene Family Members in Gossypium.

Terpene synthases (TPSs), key gatekeepers in the biosynthesis of herbivore-induced terpenes, are pivotal in the diversity of terpene chemotypes across and within plant species. Here, we constructed a gene-based pangenome of the Gossypium genus by integrating the genomes of 17 diploid and 10 tetraploid species. Within this pangenome, 208 TPS syntelog groups (SGs) were identified, comprising 2 core SGs (TPS5 and TPS42) present in all 27 analyzed genomes, 6 softcore SGs (TPS11, TPS12, TPS13, TPS35, TPS37, and TPS47) found in 25 to 26 genomes, 131 dispensable SGs identified in 2 to 24 genomes, and 69 private SGs exclusive to a single genome. The mutational load analysis of these identified TPS genes across 216 cotton accessions revealed a great number of splicing variants and complex splicing patterns. The nonsynonymous/synonymous Ka/Ks value for all 52 analyzed TPS SGs was less than one, indicating that these genes were subject to purifying selection. Of 208 TPS SGs encompassing 1795 genes, 362 genes derived from 102 SGs were identified as atypical and truncated. The structural analysis of TPS genes revealed that gene truncation is a major mechanism contributing to the formation of atypical genes. An integrated analysis of three RNA-seq datasets from cotton plants subjected to herbivore infestation highlighted nine upregulated TPSs, which included six previously characterized TPSs in G. hirsutum (AD1_TPS10, AD1_TPS12, AD1_TPS40, AD1_TPS42, AD1_TPS89, and AD1_TPS104), two private TPSs (AD1_TPS100 and AD2_TPS125), and one atypical TPS (AD2_TPS41). Also, a TPS-associated coexpression module of eight genes involved in the terpenoid biosynthesis pathway was identified in the transcriptomic data of herbivore-infested G. hirsutum. These findings will help us understand the contributions of TPS family members to interspecific terpene chemotypes within Gossypium and offer valuable resources for breeding insect-resistant cotton cultivars.

Cortexa: a comprehensive resource for studying gene expression and alternative splicing in the murine brain

BackgroundGene expression and alternative splicing are strictly regulated processes that shape brain development and determine the cellular identity of differentiated neural cell populations. Despite the availability of multiple valuable datasets, many functional implications, especially those related to alternative splicing, remain poorly understood. Moreover, neuroscientists working primarily experimentally often lack the bioinformatics expertise required to process alternative splicing data and produce meaningful and interpretable results. Notably, re-analyzing publicly available datasets and integrating them with in-house data can provide substantial novel insights. However, such analyses necessitate developing harmonized data handling and processing pipelines which in turn require considerable computational resources and in-depth bioinformatics expertise.ResultsHere, we present Cortexa—a comprehensive web portal that incorporates RNA-sequencing datasets from the mouse cerebral cortex (longitudinal or cell-specific) and the hippocampus. Cortexa facilitates understandable visualization of the expression and alternative splicing patterns of individual genes. Our platform provides SplicePCA—a tool that allows users to integrate their alternative splicing dataset and compare it to cell-specific or developmental neocortical splicing patterns. All standardized gene expression and alternative splicing datasets can be downloaded for further in-depth downstream analysis without the need for extensive preprocessing.ConclusionsCortexa provides a robust and readily available resource for unraveling the complexity of gene expression and alternative splicing regulatory processes in the mouse brain. The data portal is available at https://cortexa-rna.com/

Predicting splicing patterns from the transcription factor binding sites in the promoter with deep learning

BackgroundAlternative splicing is a pivotal mechanism of post-transcriptional modification that contributes to the transcriptome plasticity and proteome diversity in metazoan cells. Although many splicing regulations around the exon/intron regions are known, the relationship between promoter-bound transcription factors and the downstream alternative splicing largely remains unexplored.ResultsIn this study, we present computational approaches to unravel the regulatory relationship between promoter-bound transcription factor binding sites (TFBSs) and the splicing patterns. We curated a fine dataset that includes DNase I hypersensitive site sequencing and transcriptomes across fifteen human tissues from ENCODE. Specifically, we proposed different representations of TF binding context and splicing patterns to examine the associations between the promoter and downstream splicing events. While machine learning models demonstrated potential in predicting splicing patterns based on TFBS occupancies, the limitations in the generalization of predicting the splicing forms of singleton genes across diverse tissues was observed with carefully examination using different cross-validation methods. We further investigated the association between alterations in individual TFBS at promoters and shifts in exon splicing efficiency. Our results demonstrate that the convolutional neural network (CNN) models, trained on TF binding changes in the promoters, can predict the changes in splicing patterns. Furthermore, a systemic in silico substitutions analysis on the CNN models highlighted several potential splicing regulators. Notably, using empirical validation using K562 CTCFL shRNA knock-down data, we showed the significant role of CTCFL in splicing regulation.ConclusionIn conclusion, our finding highlights the potential role of promoter-bound TFBSs in influencing the regulation of downstream splicing patterns and provides insights for discovering alternative splicing regulations.

Progerin mRNA expression in non-HGPS patients is correlated with widespread shifts in transcript isoforms.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease caused primarily by a C1824T mutation in LMNA. This mutation activates a cryptic splice donor site, producing a lamin variant called progerin. Interestingly, progerin has also been detected in cells and tissues of non-HGPS patients. Here, we investigated progerin expression using publicly available RNA-seq data from non-HGPS patients in the GTEx project. We found that progerin expression is present across all tissue types in non-HGPS patients and correlated with telomere shortening in the skin. Transcriptome-wide correlation analyses suggest that the level of progerin expression is correlated with switches in gene isoform expression patterns. Differential expression analyses show that progerin expression is correlated with significant changes in genes involved in splicing regulation and mitochondrial function. Interestingly, 5' splice sites whose use is correlated with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5' splice site. Furthermore, introns whose alternative splicing correlates with progerin have reduced GC content. Our study suggests that progerin expression in non-HGPS patients is part of a global shift in splicing patterns.

Alternative splicing of CsbHLH133 regulates geraniol biosynthesis in tea plants.

Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. Additionally, it functions as an airborne signal that interacts with other members of the ecosystem. To date, the regulation of the geraniol biosynthesis in tea plants remains to be investigated. In this study, a correlation test of the content of geraniol and its glycosides with gene expression data revealed that nudix hydrolase, CsNudix26, and its transcription factor, CsbHLH133 are involved in geraniol biosynthesis. In vitro enzyme assays and metabolic analyses of genetically modified tea plants confirmed that CsNudix26 is responsible for the formation of geraniol. Yeast one-hybrid, dual-luciferase reporter, and EMSA assays were used to verify the binding of CsbHLH133 to the CsNudix26 promoter. Overexpression of CsbHLH133 in tea leaves enhanced CsNudix26 expression and geraniol accumulation, whereas CsbHLH133 silencing reduced CsNudix26 transcript levels and geraniol content. Interestingly, CsbHLH133-AS, produced by alternative splicing, was discovered and proved to be the primary transcript expressed in response to various environmental stresses. Furthermore, geraniol release was found to be affected by various factors that alter the expression patterns of CsbHLH133 and CsbHLH133-AS. Our findings indicate that distinct transcript splicing patterns of CsbHLH133 regulate geraniol biosynthesis in tea plants in response to different regulatory factors.

Splicing is dynamically regulated during limb development

Modifications to highly conserved developmental gene regulatory networks are thought to underlie morphological diversification in evolution and contribute to human congenital malformations. Relationships between gene expression and morphology have been extensively investigated in the limb, where most of the evidence for alterations to gene regulation in development consists of pre-transcriptional mechanisms that affect expression levels, such as epigenetic alterations to regulatory sequences and changes to cis-regulatory elements. Here we report evidence that alternative splicing (AS), a post-transcriptional process that modifies and diversifies mRNA transcripts, is dynamic during limb development in two mammalian species. We evaluated AS patterns in mouse (Mus musculus) and opossum (Monodelphis domestica) across the three key limb developmental stages: the ridge, bud, and paddle. Our data show that splicing patterns are dynamic over developmental time and suggest differences between the two mammalian taxa. Additionally, multiple key limb development genes, including Fgf8, are differentially spliced across the three stages in both species, with expression levels of the conserved splice variants, Fgf8a and Fgf8b, changing across developmental time. Our data demonstrates that AS is a critical mediator of mRNA diversity in limb development and provides an additional mechanism for evolutionary tweaking of gene dosage.

Impact of a worker bee thoracic ganglion RIC-3 variant on the actions of acetylcholine and neonicotinoids on nicotinic receptors in Apis mellifera.

A transmembrane thioredoxin (TMX3) enables the functional expression of insect nicotinic acetylcholine receptors (nAChRs) in Xenopus laevis oocytes, while co-factors RIC-3 and UNC-50 regulate the receptor expression level. RIC-3 (resistant to inhibitors of cholinesterase 3) has been shown to diversify by its differential mRNA splicing patterns. How such diversity influences neonicotinoid sensitivity of nAChRs of beneficial insect species remains poorly understood. We have identified a RIC-3 variant expressed most abundantly in the thoracic ganglia of honeybee (Apis mellifera) workers and investigated its effects on the functional expression and pharmacology of Amα1/Amα8/Amβ1 and Amα1/Amα2/Amα8/Amβ1 nAChRs expressed in X. laevis oocytes. The AmRIC-3 enhanced the response amplitude to the acetylcholine (ACh) of these A. mellifera nAChRs when its cRNAwas injected into oocytes at low concentrations but suppressed the ACh response amplitude at high concentrations. Co-expression of the AmRIC-3 had a minimal impact on the affinity of ACh, but changed the efficacy of imidacloprid and clothianidin, suggesting that the presence and the level of RIC-3 expression can affect the nAChR responses to ACh and neonicotinoids, depending on nAChR subunit composition in honeybees. © 2024 Society of Chemical Industry.

A CRISPR-dCas13 RNA-editing tool to study alternative splicing.

Alternative splicing allows multiple transcripts to be generated from the same gene to diversify the protein repertoire and gain new functions despite a limited coding genome. It can impact a wide spectrum of biological processes, including disease. However, its significance has long been underestimated due to limitations in dissecting the precise role of each splicing isoform in a physiological context. Furthermore, identifying key regulatory elements to correct deleterious splicing isoforms has proven equally challenging, increasing the difficulty of tackling the role of alternative splicing in cell biology. In this work, we take advantage of dCasRx, a catalytically inactive RNA targeting CRISPR-dCas13 ortholog, to efficiently switch alternative splicing patterns of endogenous transcripts without affecting overall gene expression levels cost-effectively. Additionally, we demonstrate a new application for the dCasRx splice-editing system to identify key regulatory RNA elements of specific splicing events. With this approach, we are expanding the RNA toolkit to better understand the regulatory mechanisms underlying alternative splicing and its physiological impact in various biological processes, including pathological conditions.

Enhancing novel isoform discovery: leveraging nanopore long-read sequencing and machine learning approaches.

Long-read sequencing technologies can capture entire RNA transcripts in a single sequencing read, reducing the ambiguity in constructing and quantifying transcript models in comparison to more common and earlier methods, such as short-read sequencing. Recent improvements in the accuracy of long-read sequencing technologies have expanded the scope for novel splice isoform detection and have also enabled a far more accurate reconstruction of complex splicing patterns and transcriptomes. Additionally, the incorporation and advancements of machine learning and deep learning algorithms in bioinformatic software have significantly improved the reliability of long-read sequencing transcriptomic studies. However, there is a lack of consensus on what bioinformatic tools and pipelines produce the most precise and consistent results. Thus, this review aims to discuss and compare the performance of available methods for novel isoform discovery with long-read sequencing technologies, with 25 tools being presented. Furthermore, this review intends to demonstrate the need for developing standard analytical pipelines, tools, and transcript model conventions for novel isoform discovery and transcriptomic studies.

PTBP1-mediated repression of neuron-specific CDC42 splicing constitutes a genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype glioblastoma.

Gene co-expression networks may encode hitherto inadequately recognized vulnerabilities for adult gliomas. By identifying evolutionally conserved gene co-expression modules around EGFR (EM) or PDGFRA (PM), we recently proposed an EM/PM classification scheme, which assigns IDH-wildtype glioblastomas (GBM) into the EM subtype committed in neural stem cell compartment, IDH-mutant astrocytomas and oligodendrogliomas into the PM subtype committed in early oligodendrocyte lineage. Here, we report the identification of EM/PM subtype-specific gene co-expression networks and the characterization of hub gene polypyrimidine tract-binding protein 1 (PTBP1) as a genomic alteration-independent vulnerability in IDH-wildtype GBM. Supervised by the EM/PM classification scheme, we applied weighted gene co-expression network analysis to identify subtype-specific global gene co-expression modules. These gene co-expression modules were characterized for their clinical relevance, cellular origin and conserved expression pattern during brain development. Using lentiviral vector-mediated constitutive or inducible knockdown, we characterized the effects of PTBP1 on the survival of IDH-wildtype GBM cells, which was complemented with the analysis of PTBP1-depedent splicing pattern and overexpression of splicing target neuron-specific CDC42 (CDC42-N) isoform. Transcriptomes of adult gliomas can be robustly assigned into 4 large gene co-expression modules that are prognostically relevant and are derived from either malignant cells of the EM/PM subtypes or tumor microenvironment. The EM subtype is associated with a malignant cell-intrinsic gene module involved in pre-mRNA splicing, DNA replication and damage response, and chromosome segregation, and a microenvironment-derived gene module predominantly involved in extracellular matrix organization and infiltrating immune cells. The PM subtype is associated with two malignant cell-intrinsic gene modules predominantly involved in transcriptional regulation and mRNA translation, respectively. Expression levels of these gene modules are independent prognostic factors and malignant cell-intrinsic gene modules are conserved during brain development. Focusing on the EM subtype, we identified PTBP1 as the most significant hub for the malignant cell-intrinsic gene module. PTBP1 is not altered in most glioma genomes. PTBP1 represses the conserved splicing of CDC42-N. PTBP1 knockdown or CDC42-N overexpression disrupts actin cytoskeleton dynamics, causing accumulation of reactive oxygen species and cell apoptosis. PTBP1-mediated repression of CDC42-N splicing represents a potential genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype GBM.

Circular RNAs regulate neuron size and migration of midbrain dopamine neurons during development

Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of non-coding RNAs (ncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of circular RNAs (circRNAs) at key developmental timepoints in mouse mDA neurons in vivo using fluorescence-activated cell sorting followed by short- and long-read RNA sequencing. In situ hybridisation shows expression of several circRNAs during early mDA neuron development and post-transcriptional silencing unveils roles for different circRNAs in regulating mDA neuron morphology. Finally, in utero electroporation and time-lapse imaging implicate circRmst, a circRNA with widespread morphological effects, in the migration of developing mDA neurons in vivo. Together, these data for the first time suggest a functional role for circRNAs in developing mDA neurons and characterise poorly defined aspects of mDA neuron development.

The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare)

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.

Analysis of different expression RNA binding protein genes in mouse microglia cell from the brains of mice 72 h after subarachnoid hemorrhage or sham operation

BackgroundThe prognosis of brain injury caused by subarachnoid hemorrhage (SAH) is poor. Previous studies showed that abnormal function of RBPs might be involved in brain injury, neuroinflammation and further affect microglia homeostasis. However, no studies have systematically analyzed the genome-wide abnormal expression of RBPs genes in microglia during SAH.MethodsRNA-seq data of microglia from the SAH mouse group (SAH) and control sham-operated mouse group (sham) were downloaded from the GEO database in GSE167957, including four samples from the sham group and four samples from the SAH group for subsequent analysis.Utilizing GO and KEGG functional enrichment analyses, we conducted a comprehensive study of differentially expressed genes (DEGs), alternative splicing patterns, and co-expression networks to gain deeper insights into the differential expression of RNA-binding proteins (RBPs) and differential alternative splicing events (ASEs) between the SAH (subarachnoid hemorrhage) and sham groups. This analysis aimed to elucidate the potential mechanisms underlying the aberrant expression of RBPs in microglia during brain injury caused by SAH.ResultsASEs and co-expression analyses of differentially expressed RBPs and differential ASEs were carried out in microglia in terms of gene expression. GO and KEGG functional enrichment analysis showed that aberrantly expressed RBPs such as Mcm7, Mtdh, SRSF3, and Hnrnpa2b1 may affect and regulate downstream Csnk1d, Uckl1 and other protein phosphorylation-related genes by alterative splicing.ConclusionRBPs were aberrantly expressed in microglia during the development of brain injury secondary to SAH, regulating alterative splicing of downstream genes and influencing the progression of SAH brain injury in this study. This implies that RBPs are important for the identification of new therapeutic targets for brain injury after SAH.