Post-transcriptional Splicing Research Articles

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection.

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.

CCAT1 lncRNA is chromatin-retained and post-transcriptionally spliced

Super-enhancers are unique gene expression regulators widely involved in cancer development. Spread over large DNA segments, they tend to be found next to oncogenes. The super-enhancer c-MYC locus forms long-range chromatin looping with nearby genes, which brings the enhancer and the genes into proximity, to promote gene activation. The colon cancer-associated transcript 1 (CCAT1) gene, which is part of the MYC locus, transcribes a lncRNA that is overexpressed in colon cancer cells through activation by MYC. Comparing different types of cancer cell lines using RNA fluorescence in situ hybridization (RNA FISH), we detected very prominent CCAT1 expression in HeLa cells, observed as several large CCAT1 nuclear foci. We found that dozens of CCAT1 transcripts accumulate on the gene locus, in addition to active transcription occurring from the gene. The accumulating transcripts are released from the chromatin during cell division. Examination of CCAT1 lncRNA expression patterns on the single-RNA level showed that unspliced CCAT1 transcripts are released from the gene into the nucleoplasm. Most of these unspliced transcripts were observed in proximity to the active gene but were not associated with nuclear speckles in which unspliced RNAs usually accumulate. At larger distances from the gene, the CCAT1 transcripts appeared spliced, implying that most CCAT1 transcripts undergo post-transcriptional splicing in the zone of the active gene. Finally, we show that unspliced CCAT1 transcripts can be detected in the cytoplasm during splicing inhibition, which suggests that there are several CCAT1 variants, spliced and unspliced, that the cell can recognize as suitable for export.

Post-transcriptional splicing can occur in a slow-moving zone around the gene

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Light controls mesophyll-specific post-transcriptional splicing of photoregulatory genes by AtPRMT5

Light plays a central role in plant growth and development, providing an energy source and governing various aspects of plant morphology. Previous study showed that many polyadenylated full-length RNA molecules within the nucleus contain unspliced introns (post-transcriptionally spliced introns, PTS introns), which may play a role in rapidly responding to changes in environmental signals. However, the mechanism underlying post-transcriptional regulation during initial light exposure of young, etiolated seedlings remains elusive. In this study, we used FLEP-seq2, a Nanopore-based sequencing technique, to analyze nuclear RNAs in Arabidopsis (Arabidopsis thaliana) seedlings under different light conditions and found numerous light-responsive PTS introns. We also used single-nucleus RNA sequencing (snRNA-seq) to profile transcripts in single nucleus and investigate the distribution of light-responsive PTS introns across distinct cell types. We established that light-induced PTS introns are predominant in mesophyll cells during seedling de-etiolation following exposure of etiolated seedlings to light. We further demonstrated the involvement of the splicing-related factor A. thaliana PROTEIN ARGININE METHYLTRANSFERASE 5 (AtPRMT5), working in concert with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical repressor of light signaling pathways. We showed that these two proteins orchestrate light-induced PTS events in mesophyll cells and facilitate chloroplast development, photosynthesis, and morphogenesis in response to ever-changing light conditions. These findings provide crucial insights into the intricate mechanisms underlying plant acclimation to light at the cell-type level.

Splicing under stress: A matter of time and place.

Excision of introns during splicing regulates gene expression. In this issue, work by Sung et al. (https://doi.org/10.1083/jcb.202111151) demonstrates that the timing of intron removal in response to stress is coordinated in nuclear speckles, adding a component of spatial regulation to co-/post-transcriptional splicing.

LPI-IBWA: Predicting lncRNA-protein interactions based on an improved Bi-Random walk algorithm

Many studies have shown that long-chain noncoding RNAs (lncRNAs) are involved in a variety of biological processes such as post-transcriptional gene regulation, splicing, and translation by combining with corresponding proteins. Predicting lncRNA-protein interactions is an effective approach to infer the functions of lncRNAs. The paper proposes a new computational model named LPI-IBWA. At first, LPI-IBWA uses similarity kernel fusion (SKF) to integrate various types of biological information to construct lncRNA and protein similarity networks. Then, a bounded matrix completion model and a weighted k-nearest known neighbors algorithm are utilized to update the initial sparse lncRNA-protein interaction matrix. Based on the updated lncRNA-protein interaction matrix, the lncRNA similarity network and the protein similarity network are integrated into a heterogeneous network. Finally, an improved Bi-Random walk algorithm is used to predict novel latent lncRNA-protein interactions. 5-fold cross-validation experiments on a benchmark dataset showed that the AUC and AUPR of LPI-IBWA reach 0.920 and 0.736, respectively, which are higher than those of other state-of-the-art methods. Furthermore, the experimental results of case studies on a novel dataset also illustrated that LPI-IBWA could efficiently predict potential lncRNA-protein interactions.

Identification of pararosaniline as a modifier of RNA splicing in Caenorhabditis elegans.

Posttranscriptional splicing of premessenger RNA (mRNA) is an evolutionarily conserved eukaryotic process for producing mature mRNA that is translated into proteins. Accurate splicing is necessary for normal growth and development, and aberrant splicing is increasingly evident in various human pathologies. To study environmental factors that influence RNA splicing, we employed a fluorescent Caenorhabditis elegans in vivo splicing reporter as a biomarker for splicing fidelity to screen against the US EPA ToxCast chemical library. We identified pararosaniline hydrochloride as a strong modifier of RNA splicing. Through gene expression analysis, we found that pararosaniline activates the oxidative stress response and alters the expression of key RNA splicing regulator genes. Physiological assays show that pararosaniline is deleterious to C. elegans development, reproduction, and aging. Through a targeted RNAi screen, we found that inhibiting protein translation can reverse pararosaniline's effect on the splicing reporter and provide significant protection against long-term pararosaniline toxicity. Together, this study reveals a new chemical modifier of RNA splicing and describes translation inhibition as a genetic mechanism to provide resistance.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance.

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.

Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts.

Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.

Molecular basis of inherited protein C deficiency results from genetic variations in the signal peptide and propeptide regions

BackgroundInherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC’s signal peptide and propeptide have been reported in patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear. ObjectivesTo investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC’s signal peptide and propeptide. MethodsUsing cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay. ResultsOur data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing. ConclusionOur findings suggest that variations in PC’s signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD.

RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

C27 RBM20 VARIANTS RELATED CARDIOMYOPATHY: AN ITALIAN CASE SERIES

Abstract Background RNA–binding motif protein 20 (RBM20) regulates post–transcriptional splicing, particularly in sarcomeric, but also in other cardiac genes crucial for homeostasis. RBM20 variants are a rare cause of cardiomyopathy (CMP), but genotype–phenotype correlation is still elusive, starting from the type of CMP (dilated, non–compacted, hypertrophic, and even restrictive phenotypes have been described), and ranging to sex–dependent and arrhythmic expression. Purpose: To describe cardiac involvement in 8 patients with RBM20 variants. Methods 189 patients (pts) underwent genetic analysis in our center (2019–2022) for suspected genetic CMP (TruSight Cardio panel, Illumina); 72% had a positive genetic report, including pathogenic/likely pathogenic (P/LP) or variants of undetermined significance (VUS). Among them, 9 (5%, 6 males and 3 females, from 6 families) carried a heterozygous RBM20 variant: 5 a sole P variant, 3 an RBM20–VUS associated to a second VUS in another CMP related gene, one an RBM20–VUS associated to a LP variant in BAG3; the last patient was excluded (figure with 8 pts). Results Most pts (75%) had a positive family history (FH) for sudden death. Mean age at diagnosis was significantly lower for P variant carriers compared to VUS carriers (25±15 vs 46±2 years, p=0.04); no other significant differences were found between the 2 groups. At diagnosis, 4/8 pts (50%) had NYHA class>2, mean LVEF was 34±18% and mean LV end diastolic volume 83±14 ml/m2; most pts (6/8, 75%) presented with a dilated phenotype, one with a non–compacted and one with a hypertrophic phenotype. Cardiac magnetic resonance, available in 6 pts, showed non–ischemic fibrosis in 3 (50%). At presentation, two pts had non–sustained VT (NSVT), one atrial flutter, one first degree AV block. The median follow–up was 2 years (IQR 2–10). Four pts (50%) underwent ICD implantation at 40 ± 13 years old. One female patient underwent heart transplant at the age of 13 and no one died from end stage HF; 3 pts had NSVT, one patient had atrial fibrillation (successfully treated with ablation), and none developed advanced AV blocks. Conclusions This is the first Italian case series of RMB20 variants related CMP. In our case series, RBM20 variants are associated with an early onset cardiac expression both in male and female pts, with LV dilated CMP and heart failure being the most common presentation. None of the pts presented with or developed advanced AV blocks, most suffered from NSVT.

Splicing of ultraconserved poison exon elements in RNA binding proteins reprograms the T effector immune response

Abstract Our current understanding of pathways governing the strength and longevity of T cell mediated immune responses primarily focuses on transcriptional and epigenetic changes. However, our prior work demonstrated that optimal T cell effector function is also dependent on RNA binding proteins (RBPs) that carry out post-transcriptional RNA splicing and polyadenylation. Here, we find that a specific family of RBPs, serine- and arginine-rich (SR) RBPs, are significantly upregulated by T cell activation and required for CD8 +T cell mediated responses. SR transcripts contain ultraconserved poison exon (PE) elements that are alternatively spliced and introduce premature stop codons that trigger nonsense-mediated decay. We show that differential splicing of specific RBP PE regions is both induced by TCR stimulation and required for CD8 +T cell activation in both human and mouse. Further, we demonstrate that PEs are cell-state associated by using single-cell RNA sequencing analysis of splicing changes across mouse thymic T cell subsets, in CD8 +T cells over the course of an acute Listeria infection, as well as in human lymphoblastic leukemia, atherosclerosis, and melanoma patient T cells. CRISPR-Cas9 mediated deletion of the PE element of SR-like RBP Tra2β in mouse T effector cells during influenza infection resulted in a significant increase in proliferating and surviving T cells and altered the splicing pattern of critical cell survival, proliferation, and adhesion genes. In total, our work demonstrates a fundamental role for alternatively spliced ultraconserved elements in defining T cell states, controlling T effector-mediated immunity, and altering the T cell response in pathologic conditions. This work was supported in part by NIH grants AI136955 and R21AI139891 awarded to A.T.V, institutional support, and Boehringer Ingelheim endowed Chair in Immunology.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n=6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

BackgroundAlternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.MethodsWe recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.ResultsMass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.ConclusionProteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration.

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at the Bact state, an active spliceosome but not catalytically primed, which depends on Smad Nuclear Interacting Protein 1 (SNIP1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing, and that its misregulation contributes to neurodegeneration.

Abstract PO011: Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers

Abstract Childhood liver cancers hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are rare diseases but with a rising incidence. HEMNOS (Hepatocellular malignant neoplasm, not otherwise specified) is a recent entity with histopathological features of HB and HCC. Current chemotherapy treatments are effective to shrink tumor before surgery, nonetheless can cause severe lifelong adverse effects and are not effective for patients with aggressive and metastatic HB or HCC (~20% die due to the disease). The HB and HCC diagnosis is key to assign therapeutic regimens in the ongoing Pediatric Hepatic International Tumor Trial (PHITT); however, differential diagnosis for some patients is challenging due to the lack of specific biomarkers. Nowadays, there is an urgent need to identify diagnostic and prognostic biomarkers to improve the clinical management of childhood liver cancer. Herein, we aimed to uncover the proteomic profiles of different types of childhood liver cancer and identify new biomarkers for improving tumor diagnosis and early detection of the most aggressive cases. For this, 99 frozen tissue samples from 71 patients (mean age: 54.5 months, 60.6% boys, 40.9% metastasis, 23.7% deaths) including 70 primary tumors (57 HB, 8 HCC and 5 HEM-NOS), 22 non-tumors and 7 metastases were analyzed by label free mass spectrometry analysis. A total of 5,417 proteins were sequenced in the different samples. Through supervised analysis, we identified a total of 1302 differential expressed proteins in tumors as compared to non-tumor livers (FC +2 and FDR &amp;lt;0.01). A total of 246 proteins of them (19%) were commonly differently expressed in all tumors types; having HEMNOS a mixture of HB and HCC protein profiling. A panel of potential diagnostic biomarkers including 179, 120 and 81 upregulated proteins (FC &amp;gt;2) was defined for HB, HCC and HEM-NOS, respectively. Hierarchical unsupervised clustering and principal component analysis showed two main cluster of tumors: one including HB and HEM-NOS and another one with a mixture of HB, HEM-NOS and HCC. Interestingly, this second cluster was associated with clinical and molecular features of aggressive tumors such as multifocality (p = 0.029), Epigenetic Epi-CB subclass (p = 0.006), high-risk Molecular Risk Stratification (p = 0.009) and poor survival (log rank test=0.012). A total of 2082 proteins were found to be differently expressed between the two prognostic clusters (FC +2 and FDR &amp;lt; 0.01); the GEO enrichment analysis revealed that these proteins were significantly enriched in post-transcriptional gene silencing and RNA splicing mechanisms (FDR &amp;lt;10−7). In addition, a 25-protein signature associated to liver cancer prognosis was defined (FC&amp;gt;6 and FDR &amp;lt; 10−14). In conclusion, we identified a list of potential diagnostic and prognostic protein biomarkers that after a validation in large patient cohort, could be used to improve the clinical management of childhood liver cancer. Citation Format: Álvaro Del Río-Álvarez, Juan Carrillo-Reixach, Laura Royo, Montse Domingo-Sàbat, Mikel Azkargorta, Roland Kapler, Stefano Cairo, Christian Vokuhl, Ronald de Krijger, Rita Alaggio, Marta Garrido, Gabriela Guillen, Constantino Sábado, Laura Guerra, Francisco Hernandez, Maria Elena Mateos, Manuel López-Satamaría, Barbara Torres, Maria Pilar Abad, Bajčiová Viera, Piotr Czauderna, Marie Annick Buendia, Felix Elortza, Keith Wheatley, Bruce Morland, Carolina Armengol. Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO011.