ABSTRACT: Genome-wide identification of mis-spliced/intron-retained transcripts in Arabidopsis thaliana rcf1-1 mutant plants under normal growth conditions as determined by tiling array analysis.
Project description:Cold stress is one of the most severe environmental conditions which cause huge losses in crop production worldwide. We identified a DEAD box RNA helicase, RCF1 (Regulator of CBF gene expression 1) that controls pre-mRNA splicing of cold-stress-responsive genes including positive and negative regulators of CBF genes. We used whole genome tiling array analysis under normal growth conditions to identify transcripts which are mis-spliced/intron-retained in rcf1-1.
Project description:Cold stress is one of the most severe environmental conditions which cause huge losses in crop production worldwide. We identified a DEAD box RNA helicase, RCF1 (Regulator of CBF gene expression 1) that controls pre-mRNA splicing of cold-stress-responsive genes including positive and negative regulators of CBF genes. We used whole genome tiling array analysis under cold stress to identify transcripts which are mis-spliced/intron-retained in rcf1-1.
Project description:Genome-wide identification of mis-spliced/intron-retained transcripts in Arabidopsis thaliana rcf1-1 mutant plants under cold stress as determined by tiling array analysis.
Project description:Cold stress is one of the most severe environmental conditions which cause huge losses in crop production worldwide. We identified a DEAD box RNA helicase, RCF1 (Regulator of CBF gene expression 1) that controls pre-mRNA splicing of cold-stress-responsive genes including positive and negative regulators of CBF genes. We used whole genome tiling array analysis under normal growth conditions to identify transcripts which are mis-spliced/intron-retained in rcf1-1. Fourteen-day-old wild type and rcf1-1 seedlings grown on MS agar medium (1x MS salts, 2% sucrose, 0.6% agar, pH 5.7) under normal conditions were harvested for total RNA isolation. RNA was extracted with Trizol reagent (Invitrogen) and synthesized into double-stranded DNA that was hybridized to whole genome tiling arrays (Affymetrix Arabidopsis Tiling1.0R). All hybridizations were performed with 3 biological replicates.
Project description:Cold stress is one of the most severe environmental conditions which cause huge losses in crop production worldwide. We identified a DEAD box RNA helicase, RCF1 (Regulator of CBF gene expression 1) that controls pre-mRNA splicing of cold-stress-responsive genes including positive and negative regulators of CBF genes. We used whole genome tiling array analysis under cold stress to identify transcripts which are mis-spliced/intron-retained in rcf1-1. Fourteen-day-old wild type and rcf1-1 seedlings grown on MS agar medium (1x MS salts, 2% sucrose, 0.6% agar, pH 5.7) were subjected to cold stress at 4M-BM-0C for 12 h. Total RNA was extracted with Trizol reagent (Invitrogen) and synthesized into double-stranded DNA that was hybridized to whole genome tiling arrays (Affymetrix Arabidopsis Tiling1.0R). All hybridizations were performed with 3 biological replicates.
Project description:Inactivation of Ybx1 results in RNA mis-splicing, retained intron enrichment and disruption of the transcriptional and post-translational control of extracellular signal-regulated kinase (ERK) signaling
Project description:To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. 87% of introns assayed manifest more than 50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly, or slowly, with ~3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns and introns annotated as alternative. FinallyFinally, S2 cells expressing the slow RpII215C4 mutant manifest substantially less intron retention than wild-type S2 cells. Examination of Total pA and Nascent RNA from 2 different cell populations and isolated fly heads.
Project description:Transcripts from the human and mouse CAPN10 cDNA sequences cloned into pcDNA and expressed in cultured cells undergo unexpected splicing. Peptide sequences missing from the spliced version of CAPN10 expressed in COS7 cells were determined by MS analysis.
Project description:Intron retention (IR) is an alternative splicing event where mRNAs containing unspliced introns are exported to the cytoplasm and then either translated, giving rise to new protein isoforms, or degraded via the nonsense-mediated decay pathway. However, unspliced introns are also seen in nuclear RNA. Some of these are spliced at a low rate but do eventually become fully spliced and their respective mRNAs exported, while others stay retained and are recognized by nuclear decay machineries. IR has been shown to be regulated during granulocyte and neuronal differentiation and in some cancers, and a subset of the nuclear retained introns, called detained introns, have been implicated in growth control pathways. Despite many findings on IR and its biological significance, it is still not clear whether an incompletely spliced intron observed is a true retained intron that is exported as an mRNA or is a product of slow splicing that remains in the nucleus. A better understanding is needed of the molecular events that reduce intron excision rate and determine the release of an RNA for export. We have found that some genes in mouse embryonic stem cells (mESCs) produce RNAs that are highly associated with the high molecular-weight nuclear pellet (chromatin), rather than the soluble nucleoplasm or cytoplasm. This chromatin-associated RNA is polyadenylated, usually contains one or more incompletely spliced introns, and is often extensively bound by polypyrimidine tract-binding protein 1 (PTBP1). We hypothesize that PTBP1 inhibits splicing of these introns, and this causes sequestration of the transcript on chromatin. We are studying the role of PTBP1 in inhibiting intron excision and anchoring RNAs in the nuclear and chromatin compartments of mESCs.
Project description:Minor intron-containing genes (MIGs) are crucial cell cycle regulators and are essential for cell survival. Thus, we explored whether MIGs play a role in cancer progression and cancer therapy resistance. Here we show that MIG-expression segregates sensitive and resistant prostate cancer cells from each other and them from benign tissue. Moreover, the rate of minor intron splicing, a crucial regulatory node for MIG-expression, was more efficient in patients with advanced prostate cancer. We show that the increased minor spliceosome (MiS) activity is downstream of the androgen receptor signaling axis in both therapy sensitive and insensitive prostate cancer types. The increased MiS activity was in line with the elevated expression of U6atac snRNA, a crucial MiS component. We propose the expression of U6atac as a potential point of vulnerability in cancer. MIGs are uniquely susceptible to the MiS, so we used siRNA against U6atac, which like protein-coding transcripts, is downregulated. Inhibition of MiS was sufficient to lower the tumor burden significantly in therapy-resistant (prostate) cancer cells and PCa patient-derived organoids. KD cells displayed elevated minor intron retention and alternative splicing across minor introns of MIGs which enriched for MAPK activity, DNA repair and cell cycle regulation. Importantly, these findings were confirmed by MassSpec analysis. Collectively our study nominates the MiS as a driver of highly proliferative, rapidly dividing (prostate) cancer cells that bears strong potential as a therapeutic target for many different cancer types.
