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:Ribosomal RNAs (rRNAs) biogenesis are multistep processes requiring the activity of several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remains obscure. Here, using loss-of-function approaches in mouse embryonic stem cells and deep sequencing of rRNA intermediates, we investigate at nucleotide resolution the requirements of exonucleases known to be involved in 5.8S maturation, and explore the role of the Perlman syndrome-associated 3’-5’ exonuclease Dis3l2 in rRNA processing. We expand the repertoire of Dis3l2 targets to three 5.8S, 5S, and 28S rRNAs. We uncover a novel cytoplasmic intermediate that we name ‘7SB’ rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide level insight into the exonuclease requirements for mammalian rRNA processing.
Project description:Yeast large ribosomal subunit (LSU) precursors are subject to substantial changes in protein composition during their maturation due to coordinated transient interactions with a large number of ribosome biogenesis factors and due to the assembly of ribosomal proteins. These compositional changes go along with stepwise processing of LSU rRNA precursors and with specific rRNA folding events, as revealed by recent cryo-electron microscopy analyses of late nuclear and cytoplasmic LSU precursors. Here we aimed to analyze changes in the spatial rRNA surrounding of selected ribosomal proteins during yeast LSU maturation. For this we combined a recently developed tethered tertiary structure probing approach with both targeted and high-throughput readout strategies. Several structural features of late LSU precursors were faithfully detected by this procedure. In addition, the obtained data let us suggest that early rRNA precursor processing events are accompanied by a global transition from a flexible to a spatially restricted rRNA conformation. For intermediate LSU precursors, a number of structural hallmarks could be addressed which include the fold of the internal transcribed spacer between 5.8S rRNA and 25S rRNA, the orientation of the central protuberance and the spatial organization of the interface between LSU rRNA domains I and III.
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: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 eukaryotic ribosome biogenesis is a highly orchestrated multistep process that starts at the nucleolus with the transcription of pre-rRNAs 5S and 35S. The latter comprises the mature 18S, 5.8S and 25S rRNAs separated by internal transcribed spacers (ITS1 and ITS2) and externally flanked by the 5’ETS and 3’ETS. The 35S pre-rRNA undergoes several co- and post-transcriptional processing events, which will enable the pre-60S and pre-40S particles to take independent maturation routes. Hundreds of assembly factors (AF) are required, being recruited and released hierarchically, for the proper folding of the rRNAs and correct positioning of the ribosomal proteins. One of the most intricate events that have recently been described is the removal of the ITS2-containing structure called pre-60S foot, which happens in a step-wise manner. Nop53 is an essential 60S AF that binds close to the ITS2 and plays a fundamental role in recruiting the RNA exosome for the 7S pre-rRNA processing, thereby dismantling the foot structure. Here we characterize the impact of Nop53 binding to the pre-60S on the compositional changes that happen during 60S assembly. For this purpose, preribosomes were affinity-purified with TAP-tagged 60S AFs (Nop7, Erb1, Rsa4, Arx1, Nmd3, Yvh1, and Lsg1) representative of different maturation stages both in the presence and absence of Nop53. Nop7 particles were also coimmunoprecipitated in the presence of Nop53 mutants incapable of recruiting the exosome (Nop53∆1-71, Nop53∆48-98) to compare with Nop53 depletion. The isolated preribosomes were analyzed by label-free MS/MS-based quantitative proteomics, revealing early and late-stage specific effects of Nop53 depletion.
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:Gene expression analyses of three-week old soil grown wild-type (Col-3) and apum23-1 mutant. Expression profiling was conducted as an attempt to identify the genes differentially regulated in apum23-1 mutant This study was under taken to characterize a member of pumilio family, APUM23 in Arabidopsis. Pumilio, an RNA-binding protein (RBP) containing tandem repeat PUF domains, has been known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their roles in plants are unknown. We detail the characterization of an Arabidopsis Pumilio gene, APUM23. APUM23 is constitutively expressed with higher level in metabolically active tissues, and upregulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth with serrated and scrunched leaves, abnormal venation pattern, and distorted organization of the palisade parenchyma cells, a phenotype reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on the localization, rRNA processing was examined in apum23 mutant. In apum23, 35S pre-rRNA and unprocessed 18S and 5.8S poly(A) rRNAs were accumulated without affecting steady-state levels of mature rRNAs, indicating that APUM23 participates in the degradation of rRNA by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 snoRNAs, and accumulated RNAs within nucleolus. Our results suggest that APUM23 plays an important role in plant development via rRNA processing and ribosome biogenesis. To identify the genes affected in apum23-1 as compared to wild-type (Col-3) under normal growth conditions using Arabidopsis ATH1 Genome arrays (Affymetrix). RNA samples from these plants were used to generate biotin labelled (cDNA) probes, which were then hybridized to the microarray. For data set generation, three independent samples were used, three from Col-3 and three from apum23-1 plants grown on soil for 21 days.