Splicing Mechanism Research Articles

Abstract C043: Aberrant mRNA splice variants as novel prognostic biomarkers for tumor aggressiveness and treatment resistance in African American prostate cancer

Abstract African Americans (AAs) have among the highest incidence of prostate cancer (PCa) and mortality attributable to this disease, and AA PCa disparities remains a clinical challenge. Specifically, AAs are 1.6 times more likely to develop PCa, and 2.4 times more likely to die from this disease compared to European Americans (EAs). A number of studies have shown that higher mortality and recurrence rates of PCa in AA men were observed even after adjustment for socioeconomic factors, suggesting that intrinsic biological difference may account for part of the PCa disparities. Our recent genomic study has identified global splicing events as critical drivers for promoting AA and EA PCa disparities. In this study, we utilized population-specific cell lines as in-vitro AA and EA PCa models to evaluate the molecular functions of the aberrant splice variants involved in cancer aggressiveness and drug resistance. Comprehensive functional assays (such as MTT, apoptosis, invasion, kinase activity assays, and western blotting analysis in PCa cells treated with vehicle or small molecule drugs, etc.) were employed to elucidate the functional roles of an oncogenic splice variant, PIK3CD-S, in AA and EA PCa cells in response to drug treatments. Immunohistochemistry and RNAScope assays were performed to examine the expression profile of PIK3CD-L (full length) and PIK3CD-S variants in an independent PCa cohort derived from AA and EA patients. Higher PIK3CD-S/PIK3CD-L expression ratios were detected in AA PCa cell lines and patient specimens. In addition, the functional validations have indicated that the higher PIK3CD-S/PIK3CD-L expression ratio in AA PCa cells potentially contributes to the higher cell proliferative and invasive capacities. By targeting splicing mechanism and/or PI3K isoforms using specific small molecule inhibitors in AA and EA cell models, we further explored the functional mechanisms of these oncogenic splice variants underlying the drug resistance. In conclusion, our molecular mapping/validation and functional analysis suggest that high PIK3CD-S/PIK3CD-L expression profile may contribute to the PCa aggressiveness and treatment resistance in AA PCa, potentially serving as a precision prognostic biomarker and novel drug target in advanced PCa Citation Format: Siyoung Ha, Himali Gujrati, Bi-Dar Wang. Aberrant mRNA splice variants as novel prognostic biomarkers for tumor aggressiveness and treatment resistance in African American prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C043.

CircRNA and Ageing.

Circular RNAs (circRNAs) are closed-loop RNA transcripts formed by a noncanonical back splicing mechanism. circRNAs are expressed in various tissues and cell types in a temporospatially regulated manner and have diverse molecular functions including their ability to act as miRNA sponges, transcriptional and splicing regulators, protein traps, and even templates for polypeptide synthesis. Emerging evidence suggests that circRNAs are themselves dynamically regulated throughout development in various organisms, with a substantial accumulation during ageing. Their regulatory roles in cellular pathways associated with ageing and senescence, as well as their implications in ageing-related diseases, such as neurological disease, cancer, and cardiovascular disease, suggest that circRNAs are key molecular determinants of the ageing process. Their unique structure, expression specificity, and biological functions highlight a potential capacity for use as novel biomarkers for diagnosis, prognosis, and treatment outcomes in a variety of conditions including pathological ageing. CircRNA may also have potential as target for interventions that manipulate ageing and longevity. In this chapter, we discuss the most recent advances in circRNA changes in ageing and ageing-associated disease.

Identification and Pharmacological Characterization of Two Serotonin Type 7 Receptor Isoforms from Mythimna separata

Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive molecule, as neurotransmitters regulate various biological functions in vertebrates and invertebrates by binding and activating specific 5-HT receptors. The pharmacology and tissue distribution of 5-HT receptors have been investigated in several model insects, and these receptors are recognized as potential insecticide targets. However, little is known about the pharmacological characterization of the 5-HT receptors in important agricultural pests. In this study, we investigated the sequence, pharmacology, and tissue distribution of 5-HT7 receptors from oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae), an important migratory and polyphagous pest species. We found that the 5-HT7 receptor gene encodes two molecularly distinct transcripts, Msep5-HT7L and Msep5-HT7S, by the mechanism of alternative splicing in M. separata. Msep5-HT7S differs from Msep5-HT7L based on the deletion of 95 amino acids within the third intracellular loop. Two Msep5-HT7 receptor isoforms were activated by 5-HT and synthetic agonists α-methylserotonin, 8-hydroxy-DPAT, and 5-methoxytryptamine, resulting in increased intracellular cAMP levels in a dose-dependent manner, although these agonists showed much poorer potency and efficacy than 5-HT. The maximum efficacy of 5-HT compared to the two 5-HT isoforms was equivalent, but 5-HT exhibited 2.63-fold higher potency against the Msep5-HT7S than the Msep5-HT7L receptor. These two isoforms were also blocked by the non-selective antagonist methiothepin and the selective antagonists WAY-100635, ketanserin, SB-258719, and SB-269970. Moreover, two distinct mRNA transcripts were expressed preferentially in the brain and chemosensory organs of M. separata adults, as determined by qPCR assay. This study is the first comprehensive characterization of two splicing isoforms of 5-HT7 receptors in M. separata, and the first to demonstrate that alternative splicing is also the mechanism for producing multiple 5-HT7 isoforms in insects. Pharmacological and gene expression profiles offer important information that could facilitate further exploration of their function in the central nervous system and peripheral chemosensory organs, and may even contribute to the development of new selective pesticides.

Circular Guide RNA for Improved Stability and CRISPR-Cas9 Editing Efficiency in Vitro and in Bacteria.

Due to its intrinsic RNA properties, guide RNA (gRNA) is the least stable component of the CRISPR-Cas9 complex and is a major target for modification and engineering to increase the stability of the system. While most strategies involve chemical modification and special processes, we created a more stable gRNA with an easy-to-use biological technique. Since circular RNAs are theoretically immune to all RNA exonucleases, we attempted to construct a circular gRNA (cgRNA) employing the autocatalytic splicing mechanism of the RNA cyclase ribozyme. First, the formation of the cgRNA, which has a length requirement, was optimized in vivo in E. coli cells. It was found that a cgRNA with an insert length of 251 bp, designated 251cgRNA, was functional. More importantly, cgRNA increased the editing efficiency of the tested base editors relative to normal linear gRNA. The cgRNAs were more stable in vitro under all tested temperature conditions and maintained their function for 24 h at 37 °C, while linear gRNAs completely lost their activity within 8 h. Enzymatically purified 251cgRNA demonstrated even higher stability, which was obviously presented on gels after 48 h at 37 °C, and maintained partial function. By inserting a homologous arm into the 251cgRNA to 251HAcgRNA cassette, the circularization efficiency reached 88.2%, and the half-life of 251HAcgRNA was 30 h, very similar to that of purified 251cgRNA. This work provides a simple innovative strategy to greatly increase the stability of gRNA both in vivo in E. coli and in vitro, with no additional cost or labor. We think this work is very interesting and might revolutionize the form of gRNAs people are using in research and therapeutic applications.

Single nucleotide polymorphism (SNP) in the doublesex (dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae

Background: Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex (dsx) gene, involved in mosquitos’ somatic sexually dimorphic traits determination. However, the pathways that trigger the signal of dsx gene exon skipping alternative splicing mechanism in anopheline mosquitoes are not well characterized. This study aims to screen the An. gambiae dsx gene splice site sequences for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene (Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs. Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

Tau Isoforms: Gaining Insight into MAPT Alternative Splicing

Tau microtubule-associated proteins, encoded by the MAPT gene, are mainly expressed in neurons participating in axonal transport and synaptic plasticity. Six major isoforms differentially expressed during cell development and differentiation are translated by alternative splicing of MAPT transcripts. Alterations in the expression of human Tau isoforms and their aggregation have been linked to several neurodegenerative diseases called tauopathies, including Alzheimer's disease, progressive supranuclear palsy, Pick's disease, and frontotemporal dementia with parkinsonism linked to chromosome 17. Great efforts have been dedicated in recent years to shed light on the complex regulatory mechanism of Tau splicing, with a perspective to developing new RNA-based therapies. This review summarizes the most recent contributions to the knowledge of Tau isoform expression and experimental models, highlighting the role of cis-elements and ribonucleoproteins that regulate the alternative splicing of Tau exons.

Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics

ABSTRACT Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted “reference isoform” 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.

RAS Mutations in Adult Acute Myeloid Leukemia (AML). Frequency, Mutational Spectrum, and Identification of a Comutation Bias for KRASK117 (TET2/ASXL1)

Introduction RAS mutations are subclonal events associated with both good and bad prognosis AML categories (Papaemmanuil E et al.N Engl J Med 2016; 374). An adverse prognostic meaning of RAS mutations in treated AML with myelodysplastic-related changes has also been reported.In solid tumors, it has been shown that patients with KRASQ61K do not confer resistance to therapy, unlike other mutations: KRASQ61K introduces a cryptic splice donor site and causes a short inactive isoform (Kobayashi Y et al. Nature 2022 ;603). However, most KRASQ61K patients acquired the silent mutation G60G, which removes the new splice donor site and increases therapy resistance. This splicing mechanism was not present in cases with NRASQ61K and HRASQ61K. We investigated the frequency of RAS mutations, their molecular associations, and the prevalence of splicing mechanisms involving KRASQ61K and NRASQ61K in AML. Methods We included a consecutive series of adult de novo AML enrolled in the CETLAM trials, a total of 620 patients who were diagnosed between 2017 to 2022. DNA and RNA were extracted from bone marrow or peripheral blood. Next Generation Sequencing (NGS) was carried out with three panels containing at least 42 AML-related genes, including KRAS and NRAS. Only variants with a VAF (variant allele frequency) >4% and ranked as pathogenic or likely pathogenic were used for the analysis. Conventional karyotype was routinely assessed. cDNA was obtained from 7 patients with NRASQ61K, and was amplified through a conventional PCR. cDNA products were observed in gel electrophoresis and sequenced by the Sanger method. IBM SPSS Statistics was used to perform Chi-Square tests. In statistical tests regarding molecular associations, patients with co-mutated NRAS and KRAS are excluded. Results Our study found RAS mutations in 16.9% (105 patients) of adult AML cases. 11.9% of AML patients had mutated NRAS, while 6.1% mutated KRAS (74 and 38 patients, respectively). In our study, Q61 was the third most common mutation in NRAS with an incidence of 26%, while it was the fourth most common in KRAS with a frequency of 12%. The distribution of mutations in KRAS and NRAS is shown in figure 1. Regarding the Q61 codon, the distribution of each amino acid change in our cohort is shown in Figure 1b. Nine NRASQ61K were found, and cDNA could be obtained from 7 patients. NRASQ61K and non-mutated NRAS samples presented the standard large transcript in the gel electrophoresis. These findings ruled out the presence of exon skipping in AML with NRASQ61K mutations. No single case of KRASQ61K was observed in this extensive series of AML, so we could not identify alternative transcripts described in solid tumors nor compensatory KRASG60G mutations.1.1% of patients (7 cases) presented co-mutated NRAS and KRAS. In 9 subjects, more than one NRAS mutation was found, whereas, in 3 patients we detected two KRAS mutations. Lastly, we focused on molecular associations of RAS mutated patients (Figure 2). Of the total RAS-mutated cohort (105 patients), DNMT3A and NPM1 had the highest mutation frequency. Notably, 40.3% of NRAS-mutated patients had NPM1 mutations, significantly higher (p=0.018) than KRAS (16.1%). Another interesting finding was that KRASK117N mutations were mutually exclusive with DNMT3A and NPM1. All KRASK117N (6 cases) were associated with TET2 and or ASXL1 mutations. Cytogenetics information was available for 87 patients with RAS mutations (59 NRAS and 35 KRAS). NRAS-mutated patients showed a higher frequency of core binding factor (CBF) alterations than KRAS (18.1% vs. 3.6%, respectively) (p=0.063). Complex and miscellaneous karyotypes had higher rates of KRAS mutations than NRAS mutations (57.1% vs. 32.7%, respectively, p=0.032).In 26 cases, RAS mutations were the single mutation or the one with the highest VAF; notably, 42.3% had an NRASQ61 mutation. We had cytogenetic information of 20 patients, and twelve had inv(16) (60%). Conclusions In this cohort of adult AML cases, RAS mutations were present in 16.9%. Mutations at codon G12 were the most prevalent lesions in AML. NRAS mutations were more frequent than those of KRAS. Up to 17% of RAS mutated AML cases showed more than one mutation. CBF chromosomal abnormalities were commonly associated with NRAS mutations. Complex and miscellaneous karyotypes showed KRAS mutations more often. KRASK117N mutations were consistently associated with TET2 and ASXL1 mutations and mutually exclusive with NPM1 and DNMT3A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of WNT4 alternative splicing patterns and effects on proliferation of granulosa cells in goat

Increasing ovulation numbers is one of the most important ways to promote reproduction in mammals, and follicular granulosa cells (GCs) provide the necessary nutrients and microenvironment for oocytes to ovulate. WNT4 has been shown to be a key factor in regulating the proliferation of GCs in mammalian ovarian tissues. Our previous transcriptome sequencing (RNA-seq) results have identified two alternatively spliced products of WNT4;however, little is known about the splicing mechanism and its effect on GC proliferation. In this study, two alternatively spliced products of WNT4, designated WNT4-α and WNT4-β, were identified by cloning and analyzed for their function by bioinformatics. The RT–qPCR and Western blot results showed that the expression of WNT4-α was significantly higher than that of WNT4-β in the ovary tissues and GCs of Yunshang black goats. We therefore hypothesized that WNT4-α was the main isoform affecting the proliferation of goat GCs. Subsequently, goat GC proliferation assays showed that overexpression of WNT4-α significantly promoted GC proliferation, and the opposite was true after WNT4-α inhibition. The expression of marker genes of the Wnt signaling pathway was also examined and WNT4-α was found to affect the proliferation and hormone secretion of goat GCs by regulating the Wnt signaling pathway. In addition, a series of splicing factors were involved in in the alternative splicing; in this study, SRSF6 was found to be involved as a splicing factor in the generation of WNT4 alternative splicing. In summary, WNT4 alternative splicing was mediated by the splicing factor SRSF6, and WNT4-α alternative splicing played an important role in follicle development and had a significant effect on the proliferation of goat GCs. The results of this study provide a theoretical foundation for further understanding the molecular regulatory mechanisms of the WNT4 in follicle development in goats.

Abstract A055: Altered RNA splicing causes pancreatic cancer and exposes a therapeutic vulnerability

Abstract With the aim to identify new second-hit mutations that cause pancreatic ductal adenocarcinoma (PDAC), other than mutations in p53, we performed a genetic interaction analysis based on the concept of mutually exclusive mutations, which suggests that mutations that are exclusive to one another function within the same pathway. Using our unbiased quantitative methods to evaluate the effects of somatic mutations on cancer we analyzed >3000 PDAC cases to prioritize mutations among hundreds that appear at low frequencies (5-10%) and that are mutually exclusive with mutations in p53. We found that mutations in RNA splicing factors SF3B1 and RBM10 (~15% of cases), were among the most significant, and mutually exclusive, to mutant p53. Thus, we hypothesized that aberrant RNA splicing promoted by SF3B1K700E and loss of RBM10 cause an oncogenic program that leads to tumorigenesis and therapy resistance. To test this hypothesis, first, we engineered mouse models to co-express KrasG12D with either Sf3b1K700E or loss of Rbm10 in Mist1 and Pdx1 driven expressing pancreatic cells. Interestingly, mice harboring KrasG12D and Sf3b1 K700E or loss of RBM10—like mutant p53—caused PDACs in mice. To identify the RNA splicing mechanism that leads to PDAC formation, we performed deep RNA sequencing and unbiased transcriptome-wide splicing analyses in isolated PDAC cells bearing wild-type, SF3B1K700E and loss of RBM10, derived from murine and patient PDACs. We found that like the splicing defects induced by p53mut—SF3B1K700E or loss of RBM10—induce splicing defects in sequence-specific coding exons in mRNAs derived from chromosomes 19, 7, 1 and 2 and impacting mainly GTPase activation and lipid metabolism pathways across species models. Consistent with this common mechanism, we found that PDAC cells do not tolerate the forced expression of mutant p53 with either SF3B1K700E or loss of Rbm10, supporting the mutual exclusivity findings from patient samples. To determine a personalized therapy pathway for patients bearing mutant splicing-factor tumors, we found that PDAC cells with SF3B1K700E or RBM10 loss cells are more sensitive to Gemcitabine rather than 5FU, which are the two main chemotherapeutic components of the two standard-of-care therapies. In addition, these mutations sensitize cells to small-molecule splicing inhibitor H3B-8800, which has advanced to phase 2 trial for hematologic malignancies. Lastly, we found that combination of Gemcitabine and H3B-8800 synergize to selective kill mutant splicing cells. We are currently testing these combination therapies in our genetically engineered mouse models and patient derived organoids. We are starting a phase 2 trial for Gemcitabine/Abraxane with escalating doses of H3B-8800 for tumors that have either SF3B1K700E, RBM10 loss or neomorphic p53, all which have aberrant RNA splicing. This work has determined novel drivers and mechanisms of PDAC development and a precision therapeutic strategy for the treatment of 45% pancreatic cancer patients (SF3B1K700E [5%], RBM10 loss [10%] and neomorphic p53 [30%]). Citation Format: Natasha Pinto Medici, Diana Martinez Saucedo, Danny Lee, Li-Ting Ku, Robert Tseng, Vincent Cannataro, Deanne Yugawa, Sumedha Chowdhury, Jeffrey Townsend, Christine A. Iacobuzio-Donahue, Omar Abdel-Wahab, Steven D. Leach, Luisa Escobar-Hoyos. Altered RNA splicing causes pancreatic cancer and exposes a therapeutic vulnerability [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A055.

HNRNPU promotes the progression of triple-negative breast cancer via RNA transcription and alternative splicing mechanisms

Triple-negative breast cancer (TNBC) is a great detriment to women’s health due to the lack of effective therapeutic targets. In this study, we employed an integrated genetic screen to identify a pivotal oncogenic factor, heterogeneous nuclear ribonucleoprotein U (HNRNPU), which is required for the progression of TNBC. We elucidated the pro-oncogenic role of HNRNPU, which can induce the proliferation and migration of TNBC cells via its association with DEAD box helicase 5 (DDX5) protein. Elevated levels of the HNRNPU-DDX5 complex prohibited the intron retention of minichromosome maintenance protein 10 (MCM10) pre-mRNA, decreased nonsense-mediated mRNA decay, and activated Wnt/β-catenin signalling; on the other hand, HNRNPU-DDX5 is located in the transcriptional start sites (TSS) of LIM domain only protein 4 (LMO4) and its upregulation promoted the transcription of LMO4, consequently activating PI3K-Akt-mTOR signalling. Our data highlight the synergetic effects of HNRNPU in RNA transcription and splicing in regulating cancer progression and suggest that HNRNPU may act as a potential molecular target in the treatment of TNBC.

Identification of sex-specific splicing via comparative transcriptome analysis of gonads from sea cucumber Apostichopus japonicus

Alternative splicing (AS) is an essential post-transcriptional regulation mechanism for sex differentiation and gonadal development, which has rarely been reported in marine invertebrates. Sea cucumber (Apostichopus japonicus) is an economically important marine benthic echinoderm with a potential XX/XY sex determination mechanism, whose molecular mechanism in the gonadal differentiation has not been clearly understood. In this study, we analyzed available RNA-seq datasets of male and female gonads to explore if AS mechanism exerts an essential function in sex differentiation and gonadal development of A. japonicus. In our results, a total of 20,338 AS events from 7219 alternatively spliced genes, and 189 sexually differential alternative splicing (DAS) events from 156 genes were identified in gonadal transcriptome of sea cucumber. Gene Ontology analysis indicated that these DAS genes were significantly enriched in spermatogenesis-related GO terms. Maximal Clique Centrality (MCC) was then applied for protein-protein interaction (PPI) analysis to search for protein interactions and hub DAS gene. Among all DAS genes, we identified 10 DAS genes closely related to spermatogenesis and (or) sperm motility and a hub gene dnah1. Thus, this study revealed that alternative isoforms were generated from certain genes in female and male gonads through alternative splicing, which may provide direct evidence that alternative splicing mechanisms participate in female and male gonads. These results suggested a novel perspective for explaining the molecular mechanisms underlying gonadal differentiation between male and female sea cucumbers.

MYC regulates a pan-cancer network of co-expressed oncogenic splicing factors.

MYC is dysregulated in >50% of cancers, but direct targeting of MYC has been clinically unsuccessful. Targeting downstream MYC effector pathways represents an attractive alternative. MYC regulates alternative mRNA splicing, but the mechanistic links between MYC and the splicing machinery in cancer remain underexplored. Here, we identify a network of co-expressed splicing factors (SF-modules) in MYC-active breast tumors. Of these, one is a pan-cancer SF-module correlating with MYC activity across 33 tumor types. In mammary cell models, MYC activation leads to co-upregulation of pan-cancer module SFs and to changes in >4,000 splicing events. In breast cancer organoids, co-overexpression of the pan-cancer SF-module induces MYC-regulated splicing events and increases organoid size and invasiveness, while knockdown decreases organoid size. Finally, we uncover a MYC-activity pan-cancer splicing signature correlating with survival across tumor types. Our findings provide insight into the mechanisms of MYC-regulated splicing and for the development of therapeutics for MYC-driven tumors.

The Sex-Specific Splicing of Doublesex in Brine Shrimp Artemia franciscana.

The understanding of sex determination and differentiation in animals has recently made remarkable strides through the use of advanced research tools. At the gene level, the Mab-3-related transcription factor (Dmrt) gene family, which encodes for the typical DNA-binding doublesex/Mab-3 (DM) domain in their protein, is known for its contribution to sex determination and differentiation in insects. In this study, DNA-binding DM domain screening has identified eight transcripts from Artemia franciscana transcriptomic that encode proteins containing one conserved DNA-binding DM domain. The genome mapping confirmed that these eight transcripts are transcribed from six different loci on the A. franciscana genome assembly. One of those loci, the Af.dsx-4 locus, is closely related to Doublesex, a gene belonging to the Dmrt gene family. This locus could be transcribed into three alternative transcripts, namely Af.dsx4, Af.dsxF and Af.dsxM. While Af.dsx4 and Af.dsxF could putatively be translated to form an identical Af.dsxF protein of 186 aa long, Af.dsxM translates for an Af.dsxM protein of 289 aa long but shares a DNA-binding DM domain. Interestingly, Af.dsxF and Af.dsxM are confirmed as sex-specific transcripts, Af.dsxF is only present in females, and Af.dsxM is only present in male individuals. The results suggest that the sex-specific splicing mechanism of the doublesex described in insects is also present in A. franciscana. Af.dxs-4 locus can be used in further studies to clarify the sex determination pathways in A. fracnciscana.

Human PRPF39 is an alternative splicing factor recruiting U1 snRNP to weak 5' splice sites.

Human PRPF39 is a homolog of the yeast Prp39 and Prp42 paralogs. We have previously shown that human PRPF39 forms a homodimer that interacts with the CTD of U1C, mirroring the yeast Prp39/Prp42 heterodimer. We demonstrate here that PRPF39 knockdown in HEK293 cells affects many alternative splicing events primarily by reducing the usage of weak 5'ss. Additionally, PRPF39 preferentially binds to a GC-rich RNA, likely at the interface between its NTD and CTD. These data indicate that PRPF39 potentially recruits U1 snRNP to a weak 5' ss, serving as a previously unrecognized alternative splicing factor. We further demonstrate that human TIA1 binds to U1C through its RRM1 and RRM3+Q domains but has no significant binding to PRPF39. Finally, all three human LUC7L isoforms directly interact with U1C. These results reveal significant parallels to the yeast U1 snRNP structure and support the use of yeast U1 snRNP as a model for understanding the mechanism of human alternative splicing.

New insights into the characteristics of DRAK2 and its role in apoptosis: From molecular mechanisms to clinically applied potential.

As a member of the death-associated protein kinase (DAPK) family, DAP kinase-associated apoptosis-inducing kinase 2 (DRAK2) performs apoptosis-related functions. Compelling evidence suggests that DRAK2 is involved in regulating the activation of T lymphocytes as well as pancreatic β-cell apoptosis in type I diabetes. In addition, DRAK2 has been shown to be involved in the development of related tumor and non-tumor diseases through a variety of mechanisms, including exacerbation of alcoholic fatty liver disease (NAFLD) through SRSF6-associated RNA selective splicing mechanism, regulation of chronic lymphocytic leukemia and acute myeloid leukemia, and progression of colorectal cancer. This review focuses on the structure, function, and upstream pathways of DRAK2 and discusses the potential and challenges associated with the clinical application of DRAK2-based small-molecule inhibitors, with the aim of advancing DRAK2 research.

CASA: a comprehensive database resource for the COVID-19 Alternative Splicing Atlas

BackgroundAs a key process in transcriptional regulatory mechanisms, alternative splicing (AS) plays a crucial role in maintaining the diversity of RNA and protein expression, and mediates the immune response in infectious diseases, especially for the COVID-19. Therefore, urgent data gathering and more research of AS profiles in microbe-infected human cells are needed to improve understanding of COVID-19 and related infectious diseases. Herein, we have created CASA, the COVID-19 Alternative Splicing Atlas to provide a convenient computing platform for studies of AS in COVID-19 and COVID-19-related infectious diseases.MethodsIn CASA, we reanalyzed thousands of RNA-seq datasets generated from 65 different tissues, organoids and cell lines to systematically obtain quantitative data on AS events under different conditions. A total of 262,994 AS events from various infectious diseases with differing severity were detected and visualized in this database. In order to explore the potential function of dynamics AS events, we performed analysis of functional annotations and drug-target interactions affected by AS in each dataset. RNA-binding proteins (RBPs), which may regulate these dynamic AS events are also provided for users in this database.ResultsCASA displays microbe-induced alterations of the host cell splicing landscape across different virus families and helps users identify condition-specific splicing patterns, as well as their potential regulators. CASA may greatly facilitate the exploration of AS profiles and novel mechanisms of host cell splicing by viral manipulation. CASA is freely available at http://www.splicedb.net/casa/.

Decrease of RNA editing in the failing heart leads to induction of circRNAs

Abstract Background and purpose Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process that affects the secondary structure of RNAs. Changes in RNA editing have been associated with human diseases. We therefore aimed to analyze editing in the healthy and failing human heart. Methods and results Transcriptome sequencing of human heart samples of heart failure (HF) patients (n=20) and controls (n=10) revealed A-to-I editing as the major type of editing (>80%). In HF patients, RNA editing was reduced, which was primarily attributable to Alu elements in introns of protein-coding genes. We identified 166 upregulated circRNAs in HF, with the majority showing reduced RNA editing in their parental host gene (88.3%). CircRNA expression did not correlate with their corresponding host gene (R=0.07, P<0.05), suggesting that an alternative splicing mechanism gives rise to the elevated circRNA levels in HF. The RNA editing enzyme ADAR2, which binds to RNA regions that are edited from adenosine to inosine, was decreased in failing human hearts (−68.2%). In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, we examined the formation of circRNAs on one exemplary candidate. AKAP13 was among the top edited mRNAs in the human heart and gave rise to a circular transcript, which was elevated in HF. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes (−31.0%). Conclusion Our study shows that ADAR2 mediates A-to-I RNA editing in the human heart. We describe an alternative splicing mechanism of circRNAs in the human heart. In the healthy human heart, A-to-I RNA editing represses the formation of dsRNA structures of Alu elements thereby favoring linear mRNA splicing. Our results contribute to a better mechanistic understanding into the human-specific regulation of circRNA formation and are relevant to diseases with reduced RNA editing and increased circRNA levels. Funding Acknowledgement Type of funding sources: None.

Characterization of circular RNAs with advanced sequencing technologies in human complex diseases.

Circular RNAs (circRNAs) are one category of non-coding RNAs that do not possess 5' caps and 3' free ends. Instead, they are derived in closed circle forms from pre-mRNAs by a non-canonical splicing mechanism named "back-splicing." CircRNAs were discovered four decades ago, initially called "scrambled exons." Compared to linear RNAs, the expression levels of circRNAs are considerably lower, and it is challenging to identify circRNAs specifically. Thus, the biological relevance of circRNAs has been underappreciated until the advancement of next generation sequencing (NGS) technology. The biological insights of circRNAs, such as their tissue-specific expression patterns, biogenesis factors, and functional effects in complex diseases, namely human cancers, have been extensively explored in the last decade. With the invention of the third generation sequencing (TGS) with longer sequencing reads and newly designed strategies to characterize full-length circRNAs, the panorama of circRNAs in human complex diseases could be further unveiled. In this review, we first introduce the history of circular RNA detection. Next, we describe widely adopted NGS-based methods and the recently established TGS-based approaches capable of characterizing circRNAs in full-length. We then summarize data resources and representative circRNA functional studies related to human complex diseases. In the last section, we reviewed computational tools and discuss the potential advantages of utilizing advanced sequencing approaches to a functional interpretation of full-length circRNAs in complex diseases. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA in Disease and Development > RNA in Disease.

Insights into established and emerging roles of SR protein family in plants and animals.

Splicing of pre-mRNA is an essential part of eukaryotic gene expression. Serine-/arginine-rich (SR) proteins are highly conserved RNA-binding proteins present in all metazoans and plants. SR proteins are involved in constitutive and alternative splicing, thereby regulating the transcriptome and proteome diversity in the organism. In addition to their role in splicing, SR proteins are also involved in mRNA export, nonsense-mediated mRNA decay, mRNA stability, and translation. Due to their pivotal roles in mRNA metabolism, SR proteins play essential roles in normal growth and development. Hence, any misregulation of this set of proteins causes developmental defects in both plants and animals. SR proteins from the animal kingdom are extensively studied for their canonical and noncanonical functions. Compared with the animal kingdom, plant genomes harbor more SR protein-encoding genes and greater diversity of SR proteins, which are probably evolved for plant-specific functions. Evidence from both plants and animals confirms the essential role of SR proteins as regulators of gene expression influencing cellular processes, developmental stages, and disease conditions. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.