Differential Alternative Splicing Events Research Articles

Translatome analysis in acute ischemic stroke: Astrocytes and microglia exhibit differences in poststroke alternative splicing of expressed transcripts.

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo-Tag-seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT-PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes.

Genome-wide identification of pan-cancer common and cancer-specific alternative splicing events in 9 types of cancer

Alternative splicing (AS) has significant clinical relevance with cancers and is a potential source of neoepitopes. In this study, RNA-seq data of 94 solid tumor and matched adjacent normal tissues from 47 clinical patients covering nine cancer types were comprehensively analyzed using SUVA developed by ourselves. The results identified highly conserved pan-cancer differential alternative splicing (DAS) events and cancer-specific DAS events in a series of tumor samples, which in turn revealed the heterogeneity of AS post-transcriptional regulation across different cancers. The co-disturbed network between spliceosome factors (SFs) and common cancer-associated DAS was further constructed, suggesting the potential possibility of the regulation of differentially expressed SFs on DAS. Finally, the common cancer-associated DAS events were fully validated using the TCGA dataset, confirming the significant correlation between cancer-associated DAS and prognosis. Briefly, our study elucidates new insights into conservatived and specific DAS in cancer, providing valuable resources for cancer therapeutic targets.

CTHRC1 modulates cell proliferation and invasion in hepatocellular carcinoma by DNA methylation

BackgroundCollagen triple helix repeat containing-1 (CTHRC1), an extracellular matrix protein, is highly expressed in hepatocellular carcinoma (HCC) and linked to poor prognosis. Nevertheless, the precise mechanism of CTHRC1 in HCC is unclear.MethodsAgena MassARRAY® Methylation Analysis assessed the methylation level of CTHRC1 in the promoter region. Functional assays were conducted to investigate the effects of CTHRC1 knockdown in Hep3B2.1 cells. RNA sequencing identified differentially expressed genes and lncRNAs associated with angiogenesis after CTHRC1 knockdown. Furthermore, differential alternative splicing (AS) and gene fusion events were analyzed using rMATS and Arriba.ResultsIn HCC cell lines, CTHRC1 was highly expressed and associated with hypomethylation. Downregulation of CTHRC1 inhibited Hep3B2.1 cell proliferation, migration, and invasion, blocked cells in the G1/S phase, and promoted apoptosis. We obtained 34 mRNAs and 7 lncRNAs differentially expressed between the NC and CTHRC1 inhibitor groups. Additionally, we found 4 angiogenesis-related mRNAs and lncRNAs significantly correlated with CTHRC1. RT-qPCR results showed that knockdown of CTHRC1 in Hep3B2.1 cells resulted in significantly aberrant expression of CXCL6, LINC02127, and AC020978.8. Moreover, the role of CTHRC1 in HCC development may be associated with events, like 12 AS events and 5 pairs of fusion genes.ConclusionsHigh expressed CTHRC1 is associated with hypomethylation and may promote HCC development, involving events like angiogenesis, alternative splicing, and gene fusion.

A plant virus differentially alters DNA methylation in two cryptic species of a hemipteran vector

Epigenetic patterns including DNA methylation are known to vary between distantly related species, but it is not clear how these patterns differ at an intraspecific level. The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae; Hemiptera), encompasses several cryptic species. These cryptic species possess highly similar genomes but exhibit substantial biological and physiological differences. B. tabaci cryptic species are invasive, highly polyphagous, and transmit an array of plant infecting single stranded DNA viruses (ssDNA) –begomoviruses. In this study, DNA methylation patterns around genes and genomic features of two prominent B. tabaci cryptic species were investigated following acquisition of a monopartite ssDNA virus –tomato yellow curl virus. The cryptic species investigated included: B (also known as Middle East Asia Minor 1) and Q (also known as Mediterranean). Genomic features, such as promoters, gene bodies, and transposable elements were assessed for methylation levels in both B and Q cryptic species. Despite overall similar trends, both cryptic species showed differences in methylation levels between these genomic features. Virus induced differentially methylated regions were associated with predominantly distinct genes in B and Q cryptic species. All differentially methylated regions were assessed for differential gene expression and alternative splicing events with and without virus acquisition. DNA methylation levels were found to have a negative correlation with differential gene expression in both B and Q cryptic species. The differentially expressed genes were further grouped into hyper- and hypomethylated clusters. These clusters included genes with implications for virus-vector interactions including immune functions and xenobiotics’ detoxification. The observed DNA methylation pattern differences within each cryptic species could, in part, explain some of the biological and physiological differences between them.

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.

Effects of Gene Alternative Splicing Events on Resistance to Cryptocaryonosis of Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker (L. crocea) is a productive species in marine aquaculture with great economic value in China. However, the sustainable development of large yellow croaker is hampered by various diseases including cryptocaryonosis caused by Cryptocaryon irritans. The genetic regulation processes for cryptocaryonosis in large yellow croaker are still unclear. In this present study, we analyzed differential alternative splicing events between a C. irritans resistance strain (RS) and a commercial strain (CS). We identified 678 differential alternative splicing (DAS) events from 453 genes in RS and 719 DAS events from 500 genes in CS. A set of genes that are specifically alternatively spliced in RS was identified including mfap5, emp1, and trim33. Further pathway analysis revealed that the specifically alternative spliced genes in RS were involved in innate immune responses through the PRR pathway and the Toll and Imd pathway, suggesting their important roles in the genetic regulation processes for cryptocaryonosis in large yellow croaker. This study would be helpful for the studies of the pathogenesis of cryptocaryonosis and dissection of C. irritans resistance for L. crocea.

Differences in alternative splicing events in the adaptive strategies of two Daphnia galeata genotypes induced by fish kairomones

BackgroundDaphnia galeata is a suitable model organism for investigating predator-induced defense. Genes and pathways exhibiting differential expression between fish kairomone-treated and untreated groups in D. galeata have been identified. However, understanding of the significance of alternative splicing, a crucial process of the regulation of gene expression in eukaryotes, to this mechanism remains limited. This study measured life-history traits and conducted short-read RNA sequencing and long-read isoform sequencing of two Korean D. galeata genotypes (KB1 and KE2) to uncover the genetic mechanism underlying their phenotypic plasticity under predation stress.ResultsKB1 exhibited strategies to enhance fertility and decrease body length when exposed to fish kairomones, while KE2 deployed an adaptive strategy to increase body length. Full-length transcriptomes from KB1 and KE2 yielded 65,736 and 57,437 transcripts, respectively, of which 32 differentially expressed transcripts (DETs) were shared under predation stress across both genotypes. Prominent DETs common to both genotypes were related to energy metabolism and the immune system. Additionally, differential alternative splicing (DAS) events were detected in both genotypes in response to fish kairomones. DAS genes shared between both genotypes may indicate their significant role in the post-transcriptional stress response to fish predation. Calpain-3, involved in digestion and nutrient absorption, was identified as a DAS gene in both genotypes when exposed to fish kairomones. In addition, the gene encoding thymosin beta, which is related to growth, was found to be a statistically significant DAS only in KB1, while that encoding ultraspiracle protein, also associated with growth, was only identified in KE2. Moreover, transcripts encoding proteins such as EGF-like domain-containing protein, vitellogenin fused with superoxide dismutase, and others were identified overlapping between DAS events and DETs and potentially elucidating their association with the observed phenotypic variation in each genotype.ConclusionsOur findings highlight the crucial role of alternative splicing in modulating transcriptome landscape under predation stress in D. galeata, emphasizing the requirement for integrating gene expression and splicing analyses in evolutionary adaptation studies.

Genome-wide identification and analysis of epithelial-mesenchymal transition-related RNA-binding proteins and alternative splicing in a human breast cancer cell line

Exploring the mechanism of breast cancer metastasis and searching for new drug therapeutic targets are still the focuses of current research. RNA-binding proteins (RBPs) may affect breast cancer metastasis by regulating alternative splicing (AS) during epithelial-mesenchymal transition (EMT). We hypothesised that during EMT development in breast cancer cells, the expression level of RBPs and the gene AS pattern in the cell were significantly changed on a genome-wide scale. Using GEO database, this study identified differentially expressed RBPs and differential AS events at different stages of EMT in breast cancer cells. By establishing the correlation network of differential RBPs and differential AS events, we found that RBM47, PCBP3, FRG1, SRP72, RBMS3 and other RBPs may regulate the AS of ITGA6, ADGRE5, TNC, COL6A3 and other cell adhesion genes. By further analysing above EMT-related RBPs and AS in breast cancer tissues in TCGA, it was found that the expression levels of ADAT2, C2orf15, SRP72, PAICS, RBMS3, APOBEC3G, NOA1, ACO1 and the AS of TNC and COL6A3 were significantly correlated with the prognosis of breast cancer patients. The expression levels of all 8 RBPs were significantly different in breast cancer tissues without metastasis compared with normal breast tissues. Conclusively, eight RBPs such as RBMS3 and AS of TNC and COL6A3 could be used as predictors of breast cancer prognosis. These findings need to be further explored as possible targets for breast cancer treatment.

Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera

BackgroundAlternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio’s SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera.ResultsWe identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA’s role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis.ConclusionsThis study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI >= |.20|, p < 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p < 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Unveiling unique alternative splicing responses to low temperature in Zoysia japonica through ZjRTD1.0, a high-quality reference transcript dataset.

Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity.This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low-temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low-temperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica.

Transcriptome-wide characterization of alternative splicing regulation in Najran wheat (Triticum aestivum) under salt stress

The process of alternative splicing (AS) has emerged as a crucial mechanism in plant responses to environmental stresses, contributing to the enhancement of the required transcriptome and proteome complexity. Despite the importance of AS, there remains a paucity of studies on the regulatory implications of AS in the responses of wheat to salt stress. In the current study, transcriptome-wide changes in AS profiles were established in roots and shoots of Najran wheat treated with 200 mM NaCl. Salt stress induced AS events increasing the complexity of the transcriptome; out of all expressed genes in all samples, 32,268 genes (22.5% of expressed genes) in the roots and 31,941 genes (23.1% of expressed genes) in the shoots were subjected to AS with 3’ Alternative splice site (A3) being the most frequent AS event and mutually exclusive exon (MX) being the least common event. Moreover, the results revealed that salt stress modulates AS patterns in a tissue-specific way where 82% of AS events were differentially expressed in either root or shoot tissues, participating in organ differentiation. In Total, 423 Differential AS events associated with cytoskeletal-related categories such as microtubule-based processes, actin filament-based movements, and cytoskeletal motor activity were identified in the roots. In contrast, 393 Differential AS events associated with biological categories related to metabolic and signalling processes such as catabolic processes, and response to gibberellin were identified in the shoots. The results presented in this study enhance our understanding of salt tolerance mechanisms in wheat and provide promising insights for future functional investigations and crop improvement efforts.

RMATS-turbo: an efficient and flexible computational tool for alternative splicing analysis of large-scale RNA-seq data.

Pre-mRNA alternative splicing is a prevalent mechanism for diversifying eukaryotic transcriptomes and proteomes. Regulated alternative splicing plays a role in many biological processes, and dysregulated alternative splicing is a feature of many human diseases. Short-read RNA sequencing (RNA-seq) is now the standard approach for transcriptome-wide analysis of alternative splicing. Since 2011, our laboratory has developed and maintained Replicate Multivariate Analysis of Transcript Splicing (rMATS), a computational tool for discovering and quantifying alternative splicing events from RNA-seq data. Here we provide a protocol for the contemporary version of rMATS, rMATS-turbo, a fast and scalable re-implementation that maintains the statistical framework and user interface of the original rMATS software, while incorporating a revamped computational workflow with a substantial improvement in speed and data storage efficiency. The rMATS-turbo software scales up to massive RNA-seq datasets with tens of thousands of samples. To illustrate the utility of rMATS-turbo, we describe two representative application scenarios. First, we describe a broadly applicable two-group comparison to identify differential alternative splicing events between two sample groups, including both annotated and novel alternative splicing events. Second, we describe a quantitative analysis of alternative splicing in a large-scale RNA-seq dataset (~1,000 samples), including the discovery of alternative splicing events associated with distinct cell states. We detail the workflow and features of rMATS-turbo that enable efficient parallel processing and analysis of large-scale RNA-seq datasets on a compute cluster. We anticipate that this protocol will help the broad user base of rMATS-turbo make the best use of this software for studying alternative splicing in diverse biological systems.

Visfatin Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy.

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation.

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

Deciphering the role of alternative splicing as a potential regulator in fat-tail development of sheep: a comprehensive RNA-seq based study

Although research on alternative splicing (AS) has been widely conducted in mammals, no study has investigated the splicing profiles of genes involved in fat-tail formation in sheep. Here, for the first time, a comprehensive study was designed to investigate the profile of AS events and their involvement in fat-tail development of sheep. In total, 45 RNA-Seq samples related to seven different studies, which have compared the fat-tailed vs thin-tailed sheep breeds, were analyzed. Two independent tools, rMATS and Whippet, along with a set of stringent filters were applied to identify differential AS (DAS) events between the breeds per each study. Only DAS events that were detected by both tools as well as in at least three datasets with the same ΔPSI trend (percent spliced in), were considered as the final high-confidence set of DAS genes. Final results revealed 130 DAS skipped exon events (69 negative and 61 positive ΔPSI) belonged to 124 genes. Functional enrichment analysis highlighted the importance of the genes in the underlying molecular mechanisms of fat metabolism. Moreover, protein–protein interaction network analysis revealed that DAS genes are significantly connected. Of DAS genes, five transcription factors were found that were enriched in the biological process associated with lipid metabolism like “Fat Cell Differentiation”. Further investigations of the findings along with a comprehensive literature review provided a reliable list of candidate genes that may potentially contribute to fat-tail formation including HSD11B1, SIRT2, STRN3 and TCF7L2. Based on the results, it can be stated that the AS patterns may have evolved, during the evolution of sheep breeds, as another layer of regulation to contribute to biological complexity by reprogramming the gene regulatory networks. This study provided the theoretical basis of the molecular mechanisms behind the sheep fat-tail development in terms of AS.

Alternative splicing events driven by altered levels of GEMIN5 undergo translation.

GEMIN5 is a multifunctional protein involved in various aspects of RNA biology, including biogenesis of snRNPs and translation control. Reduced levels of GEMIN5 confer a differential translation to selective groups of mRNAs, and biallelic variants reducing protein stability or inducing structural conformational changes are associated with neurological disorders. Here, we show that upregulation of GEMIN5 can be detrimental as it modifies the steady state of mRNAs and enhances alternative splicing (AS) events of genes involved in a broad range of cellular processes. RNA-Seq identification of the mRNAs associated with polysomes in cells with high levels of GEMIN5 revealed that a significant fraction of the differential AS events undergo translation. The association of mRNAs with polysomes was dependent on the type of AS event, being more frequent in the case of exon skipping. However, there were no major differences in the percentage of genes showing open-reading frame disruption. Importantly, differential AS events in mRNAs engaged in polysomes, eventually rendering non-functional proteins, encode factors controlling cell growth. The broad range of mRNAs comprising AS events engaged in polysomes upon GEMIN5 upregulation supports the notion that this multifunctional protein has evolved as a gene expression balancer, consistent with its dual role as a member of the SMN complex and as a modulator of protein synthesis, ultimately impinging on cell homoeostasis.

Effects of miR-722 on gene expression and alternative splicing in the liver of half-smooth tongue sole after infection with Vibrio anguillarum

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture.

Accelerated Progression in Lower-Risk Myelodysplastic Neoplasms: Attenuated Cell Cycle, DNA Damage Response, and Augmented Oncogenic Transcriptome Signatures

Patients with myelodysplastic neoplasms (MDS) face the risk of transformation to acute myeloid leukemia (AML). Although prognostic scoring systems exist for risk stratification and treatment decision making in MDS patients (Garcia-Manero G. Am J Hematol. 2023;98(8):1307-1325), disease management remains challenging due to the heterogeneity of clinical courses and long-term outcomes. The natural history of patients with lower-risk MDS (LR-MDS) is very heterogeneous and some LR-MDS patients experience rapid progression despite a generally favorable prognosis. The aim of our study was to uncover the molecular mechanisms underlying accelerated progression in hematopoietic stem/progenitor cells of patients with LR-MDS regardless of driver mutations. We focused on the transcriptome of bone marrow (BM) CD34 + cells from diagnostic samples using RNA-seq and used various bioinformatic pipelines to examine differentially expressed protein-coding genes, and long non-coding RNAs (lncRNAs) as well as differential alternative splicing events. RNA-seq dataset of CD34 + ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS) showed 845 differentially expressed genes (ІlogFCІ > 1, FDR < 0.01) between these groups. CD34 + cells of prMDS exhibited a transcriptional pattern of quiescent-like cell state with overall decreased metabolism signatures and significantly reduced lineage differentiation compared to stMDS CD34 + cells. Gene set enrichment analysis (GSEA) showed that cell cycle- and cell cycle checkpoint- associated genes and activation of ATR in response to replication stress were all significantly under-represented in prMDS CD34 + BM (Fig. 1A). Cellular pathways from GO Biological Processes associated with cellular responses to stress and DNA damage responses (DDR) were downregulated in prMDS compared to stMDS. The only GO terms that were significantly upregulated in prMDS were processes related to cell-matrix adhesion or cell-cell adhesion. Indeed, key transcription factor controlling changes in cell-cell adhesion, ZEB1, was significantly overexpressed in prMDS BM CD34 + cells, whereas E-cadherin expression ( CDH1) was decreased; the expression levels of CDH1 and ZEB1 showed a significant moderate negative correlation (Fig. 1B). Additionally, prMDS samples exhibited high levels of aberrant splicing and global lncRNA expression. Analysis of aberrant transcripts, deregulated lncRNAs and their targets suggested their contribution to the attenuation of DDR pathways in prMDS. Given that the downregulation of DDR gene expression was the major feature distinguishing the prMDS CD34 + cell transcriptome from that of stMDS in our cohort, we selected - from the transcriptional profile of the top 50 downregulated genes in GSEA - a prognostic DDR-associated gene signature consisting of 19 genes. Using regularized Cox regression analysis of the expression profile of 19 DDR-associated genes, the probability of a progression-free disease was calculated in our cohort. High DDR gene expressors had a significantly higher probability of progression-free disease (p < 0.001) compared to low DDR gene expressors (Fig. 2A). Among the top 50 upregulated genes in the prMDS vs. stMDS samples, ranked by GSEA, overexpression of previously identified markers of leukemic progression and/or poor MDS/AML survival ( SPNS2, MN1, DOCK1 and others) was demonstrated. Interestingly, overexpression of NEK3, a gene not previously associated with MDS/AML progression, showed a predictive power for accelerated progression in LR-MDS with the best significance of curve separation among the genes tested (Fig. 2B). Furthermore, NEK3 expression was an independent prognostic factor (p<0.001) for progression-free disease in a multivariate analysis of important clinical and genetic factors. To conclude, our data suggest that DDR gene expression signature and NEK3 expression appear to be a significant predictor of LR-MDS progression and may be applicable at the time of diagnosis for the prognostic stratification of patients with LR-MDS, regardless of their mutational status. Supported by AZV (NU21-03-00565) and (NU23-03-00401); MH CZ-DRO (UHKT, 00023736); Program EXCELES, ID Project No. LX22NPO5102, and UP Young Researcher Grant Competition, ID Project JG_2023_016.

Transcriptome Analysis of Ovarian Tissues Reveals Key Genes Involved in the Prolificacy of Ussuri Raccoon Dog

Background: Ussuri raccoon dog is an important economic animal, which brings considerable economic benefits to Hebei, China. The high fertility rate of Ussuri raccoon dog is vital for its breeding industry. However, the molecular mechanism of prolificacy traits in raccoon dog remains unclear. Therefore, it is of great importance to identify the related genes and analyze the genetic mechanism of its high reproductive traits. Methods: In our study, RNA-seq was performed using ovarian tissues from six Ussuri raccoon dogs during estrus. The samples were divided into two groups based on the litter size over the last three consecutive years. We performed a standard transcriptomic analysis procedure to dig for differentially expressed genes (DEGs) and analyze gene function in detail. Result: Statistical analysis revealed a total of 612 DEGs between the low-fecundity and high-fecundity group including 277 upregulated and 335 downregulated DEGs, respectively. KEGG pathway analysis shows the DEGs enriched in several reproductive related pathways, such as the Circadian entrainment pathway. A total of 8 DEGs were identified with differential alternative splicing (DAS) events and recognized as DAS-DE genes. Among the up-regulated DAS-DE genes, ABCA6 and GPAM were found to be the most important genes regulating the prolificacy trait of Ussuri raccoon dog. The DEGs and DAS-DE genes identified in this study provide a probable genetic mechanism for improving prolificacy of Ussuri raccoon dog breeding.