Exonuclease Requirements for Mammalian Ribosomal RNA Biogenesis and Surveillance
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE129734 | GEO | 2019/04/13
REPOSITORIES: GEO
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Project description:The RNA exosome is an essential 3’ to 5’ processing exoribonuclease complex that mediates degradation, processing, and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a 9-subunit core and a distributive 3ʹ to 5ʹ exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-ribosomal RNA (pre-rRNA). However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other exosome subunits. Instead, it mediates EXOSC10 SUMOylation at Lys (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10, indicating that EXOSC10 SUMOylation is critical for the exosome function in rRNA processing. Furthermore, USP36 itself is a rRNA-binding protein that associates pre-rRNA. These results suggest that USP36 acts as a novel SUMO ligase to mediate EXOSC10 SUMOylation critical for the RNA exosome function in rRNA processing and ribosome biogenesis.
2023-07-20 | PXD031592 | Pride
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.
2021-09-09 | PXD023383 | Pride
Project description:Transcription by RNA polymerase I (RNAPI) represents the majority of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal rRNAs (rRNAs). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pausing-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.
2023-05-02 | GSE212930 | GEO
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.
2017-12-18 | GSE96946 | GEO
Project description:The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of pre-snRNAs during their cytoplasmic maturation. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis. To investigate DIS3L2 functions genome-wide, total RNA samples were collected from model cell lines producing either WT or mut DIS3L2 three days after induction with doxycycline. The RNA samples were rRNA-depleted before preparation of strand-specific total RNA libraries according to the standard TruSeq (Illumina) protocol. TruSeq library preparation favours RNA molecules longer than 200 nt, and shorter transcripts are suboptimal for sequencing via this protocol. Thus, to obtain information about potential DIS3L2 RNA substrates with lengths between 20 and 220 nt, another RNA-Seq was carried out in parallel (with size selection through gel purification). The stable inducible HEK293 cell lines producing DIS3L2 variants were obtained using âpAL_01â and âpAL_02â plasmid constructs and the Flp-In⢠T-REx⢠system according to the manufacturerâs guidelines. âpAL_01â and âpAL_02â plasmids are vectors for co-expression of recoded C-terminal FLAG-tagged DIS3L2 [wild type (WT) variant or its catalytic mutant counterpart (mut), respectively] and sh-miRNAs directed against endogenous DIS3L2 mRNA.
2016-07-19 | E-GEOD-82336 | biostudies-arrayexpress
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.
2011-03-01 | E-GEOD-23940 | biostudies-arrayexpress
Project description:The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies have identified multiple pathways for RNA substrates into the exosome complex in vitro, but determining which pathways are preferentially followed by individual RNA species in vivo remains challenging. Here we attempted to address this important question by an RNase protection approach. We applied an in vivo RNA-protein crosslinking technique to the exosome component Rrp44/Dis2, which has both endonuclease and exonuclease activity. The resulting high-throughput sequence reads were stratified by length of the cDNA. This should reflect the length of the RNA fragments, and therefore the RNA region that was protected by exosome binding. Striking effects were seen for short fragments, which presumably arise from RNA species that follow the “direct access” route to the Rrp44 exonuclease active site. These were strongly elevated in strains lacking exonuclease activity and, surprisingly, suppressed by point mutations in the endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggest that they are generated by the endonuclease activity. Confident mapping of the short reads is challenging but it seems clear that they are derived from only a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.
2017-02-15 | GSE94889 | GEO
Project description:The biogenesis of rRNA drives cell growth and proliferation, but mechanisms that modulate rRNA production remain poorly understood. We have conducted a genetic screen to look for factors that negatively regulate endo-siRNA generation in C. elegans, and identified the suppressor of siRNA (susi)-1and a new class of triphosphorylated antisense ribosomal siRNAs (risiRNAs). In susi-1 mutant, we observed an accumulation of siRNA sequences complementary to 18S and 26S rRNAs. risiRNAs are similar to 22G-RNA, as they are 22 nt in length and have a triphosphorylated guanidine at their 5'-end. The generation of risiRNAs depends on RNA-dependent RNA polymerases, but not the ERI/Dicer complex. risiRNAs act through the nuclear RNAi pathway and guide the Argonaute protein NRDE-3 to the nucleoli, which leads to a downregulation of pre-rRNA expression. Interestingly, low temperature and ultraviolet irradiation also induces the accumulation of risiRNA, suggesting potential regulatory functions. Cloned SUSI-1 shows homology to Dis3-like exonuclease 2 (Dis3L2) in other organisms, a gene identified as a key exonuclease involved in the 3' to 5' degradation of oligouridylated RNAs. We observed an accumulation of 3'-tail oligouridylated 26S rRNA in susi-1 mutant and in low temperature-treated animals. Consistently, the injection of 3'-tail oligouridylated 26S rRNA elicited a nuclear accumulation of NRDE-3. Our findings thus establish SUSI-1(ceDis3L2) as a suppressor of endo-siRNA generation, identify a novel class of risiRNAs that suppress pre-rRNA expression, and suggest a potential disease mechanism.
2016-12-07 | GSE90927 | GEO
Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
2017-10-04 | GSE83821 | GEO
Project description:Ribosome biogenesis is a multi-step process, during which mistakes could take place at any step of pre-rRNA processing, modification, and assembly of ribosomes. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNA, we conducted both forward and reverse genetic screenings to search for more susi mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of 26S rRNA. C27F2.4(ceBUD23) is a m7G methyltransferase of 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A methyltransferase of 18S rRNA . Mutation of these genes led to modification deficiency of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasm to nuclear and nucleolar translocation of an Argonaute protein NRDE-3. The processing deficiency of rRNAs resulted in the accumulation of risiRNAs as well. We isolated SUSI-3(RIOK-1) which is similar to human RIOK1 that cleaves 20S to 18S rRNA. We further utilized RNAi and CRISPR/Cas9 technologies to perform candidate-based reverse genetic screening and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain the homeostasis of rRNAs.
2018-05-02 | GSE113905 | GEO