ABSTRACT: 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 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 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: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: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:Hight throughput techniques have revealed huge complexity in antisense RNAs in many organisms. We have explored the complexity of this class of transcripts and functional links to Polycomb silencing thought analysis of non-coding RNAs of Arabidopsis FLC. FLC is repressed and epigenetically silenced by prolonged cold, enabling plants to undergo the floral transition. Single nucleotide resolution tiling array revealed long non-coding transcripts covering the entire FLC locus. The most abundant of these are capped and polyadenylated, initiate over a 100 nucleotide window just downstream of the sense polyA site, are differentially spliced and terminate either within the sense gene or its promoter. Their levels correlate with FLC sense transcripts in all mutants and conditions tested except cold treatment. The antisense transcripts were strongly but transiently cold-induced, much earlier than other vernalization markers, and this coincided with reduction in sense FLC transcription but not sense FLC mRNA levels. Addition of the FLC antisense 5'/sense 3' region to a GFP transgene was sufficient to confer cold-induced silencing of thet fusion; however this silencing was not epigenetically manteined. These processes were all independent of the function of the Polycomb proteins required for maintenance of FLC silencing. Our data suggest that FLC antisense transcripts induce transient FLC transcriptional silencing, possibly through promoter interference, with the epigenetic silencing requiring subsequent recruitment of Polycomb machinery.
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:Hight throughput techniques have revealed huge complexity in antisense RNAs in many organisms. We have explored the complexity of this class of transcripts and functional links to Polycomb silencing thought analysis of non-coding RNAs of Arabidopsis FLC. FLC is repressed and epigenetically silenced by prolonged cold, enabling plants to undergo the floral transition. Single nucleotide resolution tiling array revealed long non-coding transcripts covering the entire FLC locus. The most abundant of these are capped and polyadenylated, initiate over a 100 nucleotide window just downstream of the sense polyA site, are differentially spliced and terminate either within the sense gene or its promoter. Their levels correlate with FLC sense transcripts in all mutants and conditions tested except cold treatment. The antisense transcripts were strongly but transiently cold-induced, much earlier than other vernalization markers, and this coincided with reduction in sense FLC transcription but not sense FLC mRNA levels. Addition of the FLC antisense 5'/sense 3' region to a GFP transgene was sufficient to confer cold-induced silencing of thet fusion; however this silencing was not epigenetically manteined. These processes were all independent of the function of the Polycomb proteins required for maintenance of FLC silencing. Our data suggest that FLC antisense transcripts induce transient FLC transcriptional silencing, possibly through promoter interference, with the epigenetic silencing requiring subsequent recruitment of Polycomb machinery. Total seedling RNA from different genotypes and different conditions: WT, FRIGIDA, 35s::FCA (giving overexpression of FCA), FRIGIDA + 2 weeks cold, FRIGIDA + 2 weeks cold + 7 days warm.
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.
