Alternative Splicing Research Articles

Aberrant alternative splicing in cancer: Splicing events and their regulatory mechanisms (Review).

Alternative RNA splicing provides cells with transcriptomic and proteomic diversity by adding another layer of regulation to gene expression. Accumulating evidence has revealed that defects in alternative splicing contribute to a variety of features of cancer development, including the modulation of cancer heterogeneity, evasion of apoptosis of cancer cells, rewiring cancer metabolism and facilitating cancer metastasis via fine‑tuning the epithelial‑to‑mesenchymal transition process. In this review, well‑known aberrant alternative splicing events associated with multiple aspects of cancer progression were presented based on available data obtained from an extensive literature search used to construct splicing regulatory networks for each of these events. The study aims to provide a more comprehensive understanding of cancer‑associated splicing networks and more precise guidance for targeting these events for cancer treatment.

RBM25 depletion suppresses the growth of colon cancer cells through regulating alternative splicing of MNK2.

Increasing evidence suggests that deregulated RNA splicing factors play critical roles in tumorigenesis; however, their specific involvement in colon cancer remains largely unknown. Here we report that the splicing factor RBM25 is overexpressed in colon cancer, and this increased expression correlates with a poor prognosis of patients with colon cancer. Functionally, RBM25 ablation suppresses the growth of colon cancer cells both in vitro and in vivo. Mechanistically, our transcriptome-wide analysis of splicing events revealed that RBM25 regulates a large number of cancer-related alternative splicing events across the human genome in colon cancer. Particularly, RBM25 regulates the splicing of MNK2 by interacting with the poly G rich region in exon 14a, thereby inhibiting the selection of the proximal 3' splice site (ss), resulting in the production of the oncogenic short isoform, MNK2b. Knockdown of RBM25 leads to an increase in the MNK2a isoform and a decrease in the MNK2b isoform. Importantly, re-expression of MNK2b or blocking the 3' ss of the alternative exon 14a with ASO partially reverses the RBM25 knockdown mediated tumor suppression. Moreover, MNK2b levels were significantly increased in colon cancer tissues, which is positively correlated with the expression level of RBM25. Collectively, our findings uncover the critical role of RBM25 as a key splicing factor in colon cancer, suggesting its potential as a prognostic marker and therapeutic target.

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.

Abstract Tu058: Reverse Remodeling with LVAD Therapy is Associated with Alternative Splicing of CAMK2D

Therapies for end-stage heart failure are lacking and the number of patients awaiting heart transplants far outweighs availability. Left ventricular assist devices (LVADs) take over cardiac pump function and are used as a bridge-to-transplant. Notably, some patients on LVADs experience recovery of cardiac function and reversal of pathological cardiac remodeling; however, the molecular mechanisms underlying these favorable outcomes are unknown. In this study, we studied 19 paired pre- and post-LVAD samples with 10 clinically categorized as responders and 9 as non-responders. Bulk mRNA sequencing identified 486 differentially expressed genes post-LVAD, including reduced pro-inflammatory gene expression and increased expression of genes involved in metal ion binding; however, just 23 total genes were differentially expressed between responders and non-responders. To determine whether LVAD differentially impacted the proteome in responders and non-responders, we next performed quantitative proteomics on post-LVAD samples. This analysis identified 154 differentially expressed proteins and revealed that factors involved in mRNA processing were higher expressed in LVAD responders. Re-analysis of our mRNA sequencing data for gene isoforms identified that alternative splicing of calcium and calmodulin-dependent protein kinase 2-delta ( CAMK2D ) was the most significantly impacted event with non-responders having increased inclusion of exon 14 (denoting the B isoform), which encodes a nuclear localization sequence. Notably, there was a strong negative correlation between exon 14 inclusion and ΔLVEF post-LVAD. We validated these findings using targeted exon-specific quantitative PCR, which confirmed that expression of CAMK2D B was higher both pre- and post-LVAD in non-responders, while responders had post-LVAD levels matching non-failing controls. We next investigated whether responders and non-responders differed at the phospho-proteome level via mass spectrometry and found three serines in CaMK2δ exon 14 were the only differentially abundant phosphosites and all increased in non-responders. Localization studies in vitro with adenoviruses expressing phospho-null or phospho-mimetic CaMK2D B constructs revealed that these phosphorylation events precluded nuclear localization. In conclusion, we identified differential alternative splicing and phosphorylation of CaMK2δ in LVAD responders, which may represent novel therapeutic targets for heart failure recovery.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

RNA splicing as a biomarker and phenotypic driver of meningioma DNA methylation groups.

Advances in our understanding of the molecular biology of meningiomas have led to significant gains in the ability to predict patient prognosis and tumor recurrence and to identify novel targets for therapeutic design. Specifically, classification of meningiomas based on DNA methylation has greatly improved our ability to risk stratify patients, however new questions have arisen in terms of the underlying impact these DNA methylation signatures have on meningioma biology. This study utilizes RNA-seq data from 486 meningioma samples corresponding to three meningioma DNA methylation groups (Merlin-intact, Immune-enriched, and Hypermitotic), followed by in vitro experiments utilizing human meningioma cell lines. We identify alterations in RNA splicing between meningioma DNA methylation groups including individual splicing events that correlate with Hypermitotic meningiomas and predict tumor recurrence and overall patient prognosis and compile a set of splicing events that can accurately predict DNA methylation classification based on RNA-seq data. Furthermore, we validate these events using RT-PCR in patient samples and meningioma cell lines. Additionally, we identify alterations in RNA binding proteins and splicing factors that lie upstream of RNA splicing events, including upregulation of SRSF1 in Hypermitotic meningiomas which we show drives alternative RNA splicing changes. Finally, we design splice switching antisense oligonucleotides to target RNA splicing changes in NASP and MFF observed in Hypermitotic meningiomas, providing a rationale for RNA-based therapeutic design. RNA splicing is an important driver of meningioma phenotypes that can be useful in prognosticating patients and as a potential exploit for therapeutic vulnerabilities.

Abstract Tu134: Oncostatin M favors cardiomyocyte survival by promoting glycolysis

Introduction: Cardiomyocytes is in high demand of energy and is susceptible to low oxygen environment. Timely switch of energy metabolism after cardiac ischaemia is helpful for cardiomyocyte preservation. We previously identified a critical role of Oncostatin M (OSM) in cardiomyocyte proliferation. Since the level of OSM quickly rises in myocardium after myocardial injury, OSM may play a protective role in early phase after myocardial injury through metabolic regulation. Hypothesis: We hypothesized that Oncostatin M regulates cardiomyocyte energy metabolism to promote its resistance to hypoxia. Methods and Results: We cultivated cardiomyocytes under hypoxic condition observed that in OSM-treated group, the percentage of TUNEL positive cardiomyocytes significantly decreased compared with that in vehicle-treated group. Published RNA sequencing data from OSM-treated neonatal cardiomyocytes revealed alterations in genes related to energy metabolism. Our Seahorse metabolic stress assays demonstrated that OSM-treated cardiomyocytes exhibited higher glycolytic metabolism and lower mitochondrial respiration than vehicle-treated cells. Pyruvate kinase M2 (PKM2), among the regulatory enzymes, was significantly upregulated in OSM-treated cardiomyocytes as was detected via western-blotting. Interestingly, PKM2 has lower catalytic activity compared to its isotype PKM1, suggesting that the augmented glycolysis might be mediated by PKM2. The mutually exclusive splicing of the gene Pkm between exons 9 and 10 led to the switch between PKM1 and PKM2. qPCR results indicated a 1.5-fold increase in Pkm transcripts and a 2-fold increase in the ratio of Pkm2 to Pkm1 , suggesting that OSM concomitantly promoted Pkm transcription as well as alternative splicing of Pkm2 . Furthermore, we performed RNA interference to knock down Stat3 , the canonical downstream signal of OSM, in cardiomyocytesand observed that OSM failed to upregulate Pkm transcription. ChIP-qPCR analysis on cardiomyocyte cell line HL-1 confirmed an increased STAT3 binding signal upstream of Pkm after OSM treatment. Additionally, RIP-qPCR assays revealed a 3-fold increase in PTBP1-binding signal to Pkm transcripts in cardiomyocytes following OSM treatment. Conclusions: Our results suggest that OSM promotes glycolysis and suppresses mitochondrial respiration in cardiomyocytes by upregulates PKM2 through STAT3 mediated transcription of Pkm and PTBP1-mediated alternative splicing of Pkm transcripts.

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis

TGFβ1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFβ. Our work investigates the potential of blocking the ‘splicing in’ of EDA with antisense oligonucleotides to inhibit TGFβ1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFβ1 in human renal proximal tubule cells (PTEC).Human primary PTEC were treated with TGFβ1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFβ, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed.Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFβ1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA.Exogenous TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA. TGFβ1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFβ1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFβ, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFβ1 was confirmed by the use of a TGFβ receptor inhibitor.Our results demonstrate a critical role of FN EDA+ in a cycle of TGFβ driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

Abstract Mo054: Long Noncoding RNA LINC00667 Downregulation Leads to SCN5A Alternative Splicing and Arrhythmogenesis

Background: The voltage-gated sodium channel SCN5A, which encodes the Nav1.5 protein, is integral to the propagation of electrical signals in cardiac tissue. SCN5A expression is diminished in cardiomyopathy, significantly increasing the susceptibility to arrhythmias. This reduction in channel activity can be attributed to decreased mRNA stability coupled with aberrant mRNA splicing. The underlying mechanisms driving the dysregulation of mRNA splicing under pathophysiological conditions remain poorly understood. Objective: This study explores the role of long noncoding RNA LINC00667 in the abnormal alternative splicing of SCN5A mRNA. Methods: LINC00667 expression was analyzed using public databases, human heart tissue, iPSC-derived cardiomyocytes (iPSCMs), and cell lines. The impact of SCN5A on action potentials and sodium currents was studied in iPSCMs. LINC00667 and SCN5A expressions were measured using qRT-PCR, while Nav1.5 protein levels were assessed through Western blot. Alternative splicing of SCN5A mRNA was examined using PCR with exon-specific primers. Gene ontology and in silico analysis identified RNA binding proteins (RBPs) potentially interacting with LINC00667. RNA immunoprecipitation (RIP) assays confirmed direct binding with key splice factors. Group differences were assessed using 2-tailed Student’s t-tests, with P < 0.05 indicating significance. Results: LINC00667, found at chromosome 18p11.31, is expressed in the human heart and associated cells, including cardiomyocytes. Its levels are inversely related to arrhythmic risk, decreasing in heart failure and ischemic cardiomyopathy as well as under hypoxic conditions. Gene ontology linked RBPs to SCN5A mRNA splicing, with RBM25, RBM5, and TIA1 identified as LINC00667 interactors. LINC00667 reduction affected regions of SCN5A mRNA encompassing Exons 6, 24, and 28, which could potentially be linked to decreased function/expression or increased unfolded protein response (UPR). Abnormal RNA splicing decreased SCN5A-encoded sodium current, reduced action potential upstroke velocity, and shortened AP duration without altering resting membrane potential. Conclusion: Decreased LINC00667 releases splicing factors that result in abnormal SCN5A mRNA splicing and reduced sodium current. Since LINC00667 is reduced in cardiomyopathy, overexpression may be antiarrhythmic by preventing sodium channel downregulation.

Abstract We084: Deciphering the Molecular Code Underlining Postnatal Cardiomyocyte Maturation by an RNA Splicing Factor RBFox1

Mammalian hearts undergo major changes after birth during the perinatal period. While several extrinsic factors such as mechanical load, electrical stimulation, hormones, and nutrients have been implicated in this process, the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. Importantly, stem-cell derived cardiomyocytes commonly exhibit immaturity which is a major limiting factor for their application as disease models or cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanism of mammalian cardiomyocyte maturation has significant implications in both basic cardiac biology and cell-based therapy and disease modeling. In our recent analysis of the global transcriptome transition from neonatal to adult rat hearts, we found RNA splicing among the top changed pathways. Additionally, we found that expression of RBFox1, a cardiac enriched RNA splicing regulator, was significantly increased during the postnatal transition to adolescence. We further demonstrate that RBFox1 mediated post-transcriptional regulation has a potent effect to promote neonatal and stem-cell derived cardiomyocyte maturation. RNA-seq followed by Gene Ontology enrichment analysis showed that Rbfox1 led to differentially expressed genes enriched in cardiac contraction and conduction, sarcomere organization, K + ion import and muscle filament sliding, as well as differential isoform switches enriched in cardiac contraction and sarcomere structure. An evolutionarily conserved, temporal specific super enhancer exists upstream of cardiac Rbfox1 , which is occupied by active epigenetic markers in the postnatal period. We have identified the tissue and temporal transcriptional activity of this super enhancer in vitro and in vivo . Several binding motifs of transcription factors are predicted within it and a key DNA fragment was identified to be essential for enhancer activity. A transgenic mouse strain has been established to facilitate the tracing of myocyte maturation in vivo by constructing a reporter system driven by a 4xSuperEnhancer-Rbfox1Promoter-tdTomato construct. In conclusion, we propose a mechanism that RBFox1 is an intracellular regulator with tissue- and temporal-specific expression patterns that regulates cardiac maturation by promoting alternative splicing that fine tunes the genetic code.

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer’s disease (AD) pathology. We used long-read sequencing in combination with a novel bioinformatics tool (FICLE) to profile transcript diversity in the entorhinal cortex of female transgenic (TG) mice harboring a mutant form of human tau. Our analyses revealed hundreds of novel isoforms and identified differentially expressed transcripts – including specific isoforms of Apoe, App, Cd33, Clu, Fyn and Trem2 – associated with the development of tau pathology in TG mice. Subsequent profiling of the human cortex from AD individuals and controls revealed similar patterns of transcript diversity, including the upregulation of the dominant TREM2 isoform in AD paralleling the increased expression of the homologous transcript in TG mice. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of AD neuropathology.

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.

Overexpression of RBM4 promotes acute myeloid leukemia cell differentiation by regulating alternative splicing of TFEB

Alternative splicing (AS) is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative AML samples from the Cancer Genome Atlas (TCGA), focusing on the splicing factor RBM4 and its splicing mechanism on the target gene TFEB, which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis.

Exon-Skipping–Based Subtyping of Colorectal Cancers

The identification of colorectal cancer (CRC) molecular subtypes has prognostic and potentially diagnostic value for patients, yet reliable subtyping remains unavailable in the clinic. The current consensus molecular subtype (CMS) classification in CRCs is based on complex RNA expression patterns quantified at the gene level. The clinical application of these methods, however, is challenging due to high uncertainty of single-sample classification and associated costs. Alternative splicing, which strongly contributes to transcriptome diversity, has rarely been used for tissue type classification. Here, we present an AS-based CRC subtyping framework sensitive to differential exon use that can be adapted for clinical application. Unsupervised clustering was used to measure the strength of association between different categories of alternative splicing and CMS. To build a classifier, the ground truth for CMS labels was derived from expression data quantified at the gene level. Feature selection was achieved through bootstrapping and L1-penalized estimation. The resulting feature space was used to construct a subtype prediction framework applicable to single and multiple samples. The performance of the models was evaluated on unseen CRCs from 2 independent sources (Indivumed, n= 129; The Cancer Genome Atlas, n= 99). We developed a CRC subtype identifier based on 29 exon-skipping events that accurately classifies unseen tumors and enables more precise differentiation of subtypes characterized by distinct biological and prognostic features as compared to classifiers based on gene expression. Here, we demonstrate that a small number of exon-skipping events can reliably classify CRC subtypes using individual patient specimens in a manner suitable to clinical application.

RNA-DNA differences in variant calls from cattle tissues result in erroneous eQTLs

BackgroundAssociation testing between molecular phenotypes and genomic variants can help to understand how genotype affects phenotype. RNA sequencing provides access to molecular phenotypes such as gene expression and alternative splicing while DNA sequencing or microarray genotyping are the prevailing options to obtain genomic variants.ResultsWe genotype variants for 74 male Braunvieh cattle from both DNA (~ 13-fold coverage) and deep total RNA sequencing from testis, vas deferens, and epididymis tissue (~ 250 million reads per tissue). We show that RNA sequencing can be used to identify approximately 40% of variants (7–10 million) called from DNA sequencing, with over 80% precision. Within highly expressed coding regions, over 92% of expected variants were called with nearly 98% precision. Allele-specific expression and putative post-transcriptional modifications negatively impact variant genotyping accuracy from RNA sequencing and contribute to RNA-DNA differences. Variants called from RNA sequencing detect roughly 75% of eGenes identified using variants called from DNA sequencing, demonstrating a nearly 2-fold enrichment of eQTL variants. We observe a moderate-to-strong correlation in nominal association p-values (Spearman ρ2 ~ 0.6), although only 9% of eGenes have the same top associated variant.ConclusionsWe find hundreds of thousands of RNA-DNA differences in variants called from RNA and DNA sequencing on the same individuals. We identify several highly significant eQTL when using RNA sequencing variant genotypes which are not found with DNA sequencing variant genotypes, suggesting that using RNA sequencing variant genotypes for association testing results in an increased number of false positives. Our findings demonstrate that caution must be exercised beyond filtering for variant quality or imputation accuracy when analysing or imputing variants called from RNA sequencing.

RPL22 is a tumor suppressor in MSI-high cancers and a splicing regulator of MDM4

Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

Analysis of host factor networks during hepatitis B virus infection in primary human hepatocytes

BackgroundChronic hepatitis B virus (HBV) infection affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects.MethodsPHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7 days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qRT-PCR was performed to validate the RNA-sequencing results, ensuring consistent findings.ResultsWe observed significant alterations in the expression patterns of 149 genes from days 1 to 7 following HBV infection (R2 > 0.7, q < 0.05). Functional analysis of these genes identified RNA-binding proteins involved in mRNA metabolism and the regulation of alternative splicing during HBV infection. Results from qRT-PCR experiments and the analysis of two validation datasets suggest that RBM14 and RPL28 may serve as potential biomarkers for HBV-associated HCC.ConclusionsTranscriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.

Identification of Key Prognostic Alternative Splicing Events of Costimulatory Molecule-Related Genes in Colon Cancer.

This study aimed to explore the key alternative splicing events in costimulatory molecule-related genes in colon cancer and to determine their correlation with prognosis. Gene expression RNA-sequencing data, clinical data, and SpliceSeq data of colon cancer were obtained from The Cancer Genome Atlas. Differentially expressed alternative splicing events in genes were identified, Followed by correlation analysis of genes corresponding to differentially expressed alternative splicing events with costimulatory molecule-related genes. Survival analysis was conducted using differentially expressed alternative splicing events in these genes and a prognostic model was constructed. Functional enrichment, proteinprotein interaction network, and splicing factor analyses were performed. In total, 6504 differentially expressed alternative splicing events in 3949 genes were identified between tumor and normal tissues. Correlation analysis revealed 3499 differentially expressed alternative splicing events in 2168 costimulatory molecule-related genes. Moreover, 328 differentially expressed alternative splicing events in 288 costimulatory molecule-related genes were associated with overall survival. The prognostic models constructed using these showed considerable power in predicting survival. The ubiquitin A-52 residue ribosomal protein fusion product 1 and ribosomal protein S9 were the hub nodes in the protein-protein interaction network. Furthermore, one splicing factor, splicing factor proline and glutamine-rich, was significantly associated with patient prognosis. Four splicing factor-alternative splicing pairs were obtained from four alternative splicing events in three genes: TBC1 domain family member 8 B, complement factor H, and mitochondrial fission 1. The identified differentially expressed alternative splicing events of costimulatory molecule-related genes may be used to predict patient prognosis and immunotherapy responses in colon cancer.

32. pVACsplice: A Computational tool for predicting and prioritizing alternative splicing neoantigens

32. pVACsplice: A Computational tool for predicting and prioritizing alternative splicing neoantigens

Functional Analysis of the Channelrhodopsin Genes from the Green Algae of the White Sea Basin

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5′-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.