Project description:The Arabidopsis core exosome (Exo9) has a phosphorolytic activity due to the RRP41 subunit. The goal of this experiment was to determine the role of this intrinsic activity of Exo9 on the degradation of rRNA maturation by-products in Arabidopsis.
Project description:The Arabidopsis core exosome (Exo9) has a phosphorolytic activity due to the RRP41 subunit. The goal of this experiment was to determine the role of this intrinsic activity of Exo9 on the maturation of the 5.8S rRNA in Arabidopsis.
Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana seedlings. m5C sites were also analyzed in Arabidopsis trm4b-1 and trdmt1 T-DNA KO mutants for the RNA methyltransferases TRM4B and TRDMT1.
Project description:The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, the degradation of short lived non-coding RNAs and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degradation of distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before processing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild type cells are discarded before entering functional pathways, suggesting that kinetic competition with degradation proofreads the efficiency and accuracy of processing.
Project description:In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs were identified. We also found that PROMPTs become sources of 21-22 nt small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPT production. In addition, we found ~200 transcription start sites within 3’-UTR-encoding regions that produce long unspliced, exosome-sensitive antisense RNAs. The previously characterized non-coding (nc) RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of ncRNAs. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.
Project description:The nucleolus composes hundreds of proteins that play distinct roles in ribosomal RNA (rRNA) processing within Fibrillar Center/Dense Fibrillar Component (FC/DFC) units and ribosome assembly in Granular Component (GC). The sub-nucleolar localization of most proteins and how their unique localization facilitates rRNA processing have remained elusive. By screening 200 nucleolar candidate proteins with high-resolution, live-cell microscopy, we identified a previously undescribed sub-nucleolar compartment, named the periphery of DFC (PDFC). Among the 12 proteins identified in PDFC, URB1 (unhealthy ribosome biogenesis 1) is required for PDFC organization and proper 3' end processing of 47S pre-rRNA. URB1 binds between the 28S rRNA and the 3' external transcribed spacer (3' ETS) to ensure 3' ETS removal, which occurs at the DFC/PDFC boundary. Loss of URB1 leads to accumulation of aberrant 3' ETS-retained 32S pre-rRNA variants, which activates exosome-dependent nucleolar surveillance. This causes decreased 28S rRNA production, reduced cell proliferation and retarded mouse embryonic pre-implementation development. Furthermore, urb1 depletion results in developmental craniofacial disorder in zebrafish, which can be at least partially rescued by further depleting exosome components. Together, this study provides new insights into functional sub-nucleolar organizations, identifies a physiologically essential step in rRNA maturation and emphasizes the exosome-dependent pre-rRNA surveillance pathway.
