Project description:XRN 5′-3′ exoribonucleases play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of the Arabidopsis wild type and mutants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 but not nuclear XRN2 activity largely altered Arabidopsis mRNA decay profiles. In addition to the primary XRN4 substrates derived from decapping and microRNA-directed slicing, terminating ribosome- and exon junction complex-protected fragments produced from XRN4-mediated cytoplasmic decay also represent the most abundant decay intermediates of Arabidopsis mRNAs. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that polyadenylation cleavage frequently occurs after an adenosine. An increase in decay intermediates with 5′ ends upstream of a consensus motif in the xrn4 mutant suggests an endonucleolytic cleavage mechanism targeting the 3′ untranslated region of many Arabidopsis mRNAs. However, analysis of decay fragments stabilized in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence might rarely result in major decay intermediates. Together, the results of this study reveal major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.
Project description:Photoperiod is a circannual signal measured by biological systems to align growth and reproduction with the seasons. To understand the effect of photoperiod of gene expression in Arabidopsis thaliana in the absence of exogenous sugar under constant light intensity, we performed time course mRNA-seq analysis on 13-day old seedlings across three photoperiods with triplicates to identify photoperiod-regulated genes.
Project description:Cytoplasmic mRNA decay occurs through several pathways, but the contributions of these decay pathways to the degradation of specific mRNAs, and interactions between the pathways, are not well understood. We carried out a genome-wide analysis of mRNA decay rates using wild-type Arabidopsis and mutants with defects in mRNA decapping and SOV/DIS3L2. Decay rates and contributions of decapping and SOV to decay were estimated for 18,674 mRNAs using maximum likelihood modeling. Most mRNAs decayed by multiple pathways, few mRNAs degraded exclusively by mRNA decapping or SOV, and specific codon usage was linked to decay rates. Unexpected faster decay of transcripts in some genotypes was found to be independent of siRNAs; instead the data suggested an RNA buffering-like phenomenon in Arabidopsis, and that VCS (decapping) is essential for both this process and the decay of very unstable mRNAs.
Project description:Here we profiled small RNAs from whole cell, cytoplasmic and nuclear extracts from three-week-old Arabidopsis seedlings. We unexpectedly found that nuclear functional hc-siRNAs are predominantly present in the cytoplasm.
Project description:Cytoplasmic mRNA decay occurs through several pathways, but the contributions of these decay pathways to the degradation of specific mRNAs, and interactions between the pathways, are not well understood. We carried out a genome-wide analysis of mRNA decay rates using wild-type Arabidopsis and mutants with defects in mRNA decapping and SOV/DIS3L2. Decay rates and contributions of decapping and SOV to decay were estimated for 18,674 mRNAs using maximum likelihood modeling. Most mRNAs decayed by multiple pathways, few mRNAs degraded exclusively by mRNA decapping or SOV, and specific codon usage was linked to decay rates. Unexpected faster decay of transcripts in some genotypes was found to be independent of siRNAs; instead the data suggested an RNA buffering-like phenomenon in Arabidopsis, and that VCS (decapping) is essential for both this process and the decay of very unstable mRNAs.
Project description:Transcriptional profiling of Arabidopsis wild-type (Col0) control seedlings with corresponding mutant seedlings is performed using Aligent's Whole Arabidopsis Gene Expression Microarray (G2519F, V4, 4x44K).
Project description:Here we profiled small RNAs from whole cell, cytoplasmic and nuclear extracts from three-week-old Arabidopsis seedlings. We unexpectedly found that nuclear functional hc-siRNAs are predominantly present in the cytoplasm. Samples from Arabidopsis thaliana whole cell, cytoplasmic and nuclear extracts with 3 replicates for each. 9 samples in all.