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 cold stress 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 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 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: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: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:The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double stranded RNA binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by increased levels of pri-miRNAs. Whole genome tiling array analyses reveal that se, abh1/cbp80 and cbp20 mutants also share similar pre-mRNA splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, since introns retained in se, abh1/cbp80 and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for non-sense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1. Keywords: Tiling array analysis of RNA populations from wild type, se, abh1 and cbp20 mutants
