The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans
ABSTRACT: Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites to release the mature ribosomal RNAs, but the functions of many ribosome biogenesis factors involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between the yeast PIN domain protein Utp23 and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp23 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A) in budding yeast. At least two independent experiments were performed and analysed separately. A non-tagged yeast strain was also used as a negative control. Results: We found that yeast Utp23 UV-crosslinked in vivo to the snR30 snoRNA and to the eukaryotic-specific expansion segment 6 (ES6) in the 18S rRNA. Conclusion: According to our crosslinking data, Utp23 is perfectly positioned to coordinate release of the snR30 snoRNA from the 18S ES6 region. Overall design: Examination of targets for RNA-binding ribosome biogenesis factors in yeast cells.
Project description:The CRAC UV crosslinking technique identified numerous pre-rRNA binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30 and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to the sequence flanking A3 and to the RNA component of RNase MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural protein factors and NTPases. A key role in coordinating pre-ribosomal assembly and processing was confirmed by "Miller" chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and pre-ribosome compaction greatly reduced. Overall design: CRAC results are provided for four individual samples: Rrp5 Full-length (FL) in vivo, Rrp5 Full-length (FL) in vitro, Rrp5 NTD and Rrp5 CTD.
Project description:Box C/D snoRNAs snR4 and snR45, whose role was unknown, guide 18S rRNA acetylations by Kre33 in yeast. UV-crosslinking (CRAC) was used to determine binding sites of Kre33 on RNA. Overall design: Duplicate data-sets of cDNA reads of RNAs UV-crosslinked to Kre33-HTP in vivo using the CRAC protocol. Cells were in mid-log phase when irradiated, immediately collected by filtration and frozen
Project description:In eukaryotes, three of the four ribosomal RNAs (rRNAs), the 5.8S, 18S and 25S/28S rRNAs, are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (5S RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into pre-ribosomes. In mammals the 5S RNP is also central regulator of the homeostasis of the tumour suppressor p53. The nucleolar localisation of the 5S RNP and its assembly into pre-ribosomes is performed by a specialised complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterisation of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialised 5S RNA E loop binding module, contacts the Rpl5 protein and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in pre-ribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. Overall design: Refer to individual Sample (2). Crac results for Rpf2-HTP and control strain BY4741 are provided
Project description:In eukaryotic cells, inefficient splicing is surprisingly common and leads to degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here we uncover a mechanism by which intronic transcripts are targeted for nuclear degradation in fission yeast. Surprisingly, sequence elements within “suicidal” introns co-transcriptionally recruit the exosome adaptor Mmi1 not only to degrade unspliced precursor, but also to downregulate levels of the resulting mRNA. Under conditions permissive for fast splicing, Mmi1 is no longer recruited and negative expression regulation is relieved. This mechanism negatively regulates levels of the RNA-helicase DDX5/Dbp2 to ensure cell survival in response to stress. We propose that suicidal introns are maintained because they facilitate regulation of gene expression. We identify multiple novel Mmi1 targets including mRNAs, non-coding RNAs, and sn/snoRNAs. We suggest a general role in RNA regulation for Mmi1 beyond degradation of meiotic transcripts. Two biological replicates of CRAC experiments (Control and Mmi1-HTP). Six RNAseq datasets in total: three biological replicates of wt and delta Mmi1 strain.
Project description:Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes – UtpA and UtpB – interact with nascent pre-ribosomal RNA have so far been poorly understood. We have combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking to elucidate the essential function of both complexes. Here we show that UtpA contains a large composite RNA binding site and captures the 5´ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to key architectural sites such as an RNA duplex formed by the 5´ ETS and U3 snoRNA as well as the 3´ boundary of 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly. Overall design: 21 samples were analyzed in total from strains carrying HTP-tagged proteins from UTP-A and UTP-B complexes. Duplicate experiments were carried out.
Project description:In eukaryotes, biogenesis of ribosomes requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high resolution cryo-EM studies in Saccharomyces cerevisiae, information about the conformation of the earliest 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA associated with 90S pre-ribosomes. We focused our analyses on external (5´ETS) and internal (ITS1) transcribed spacers as well as the 18S region. We show that in the 35S pre-rRNA, the central region of the 18S is in a more open configuration compared to 20S pre-rRNA and that the central pseudoknot is not formed. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S part locally and is involved in organizing the central pseudoknot and surrounding structures. Our results demonstrate that the U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base-pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central region of 18S that is required for processing and 40S subunit function. Overall design: Two different ribosomal RNA species were probed with 1M7 This series contains 4 samples re-analyzed from GSE83821. The raw data associated with these samples in this series (GSM2856218-GSM286221) include the barcodes not present in the raw data from the original samples from GSE83821 (GSM2219115-GSM2219118).
Project description:The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. two treatements and one control
Project description:Genome wide screens identified negative genetic interactions between several cofactors of the exosome nuclease complex and the Bre5-Ubp3 ubiquitin protease complex. RNA-binding was shown for Bre5 with enrichment for sites over exon 2 of spliced pre-mRNAs and close to poly(A) sites. An inducible splicing-reporter showed a requirement for Bre5 in efficient in vivo splicing and for normal RNAPII elongation, specifically on splicing-competent genes. A Bre5-Ubp3 sensitive site of RNAPII ubiquitination was mapped at Lys1246 at the entrance to the active site of the large subunit. Ubiquitinated RNAPII was depleted at the TSS but enriched at the 5’ end of exon 2 and upstream of poly(A) sites, similar to Bre5. Mutation of Lys1246 reduced RNAPII occupancy upstream of the poly(A) site, consistent with reduced pausing at a potential surveillance site, but increased RNAPII residence downstream of the poly(A) site. Strains expressing RNAPII with the Lys1246 mutation showed increased levels of unspliced but poly(A)+ RNA, indicating reduced cotranscriptional splicing efficiency. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript. Overall design: RNA binding by Bre5 and Rpo21 was analysed by CRAC in S. cerevisiae. Bre5 full length protein carried a C-terminal HTP tag, in the wild-type background, in the absence of Ubp3 (ubp3∆), or in an rsp5-3 temperature sensitive strain. Bre5-HTP was analysed using a standard CRAC procedure (Granneman et al., 2009). Rpo21 full length protein also carried a C-terminal HTP tag, in the wild-type background or in the absence of Bre5 (bre5∆). To map the locations of RNAPII containing ubiquitinated Rpo21 we applied modification CRAC (mCRAC), including an affinity purification step to specifically enrich for ubiquitinated proteins using a MultiDsk construct (MD), comprised of five UBA domains from the yeast ubiquitin-binding protein Dsk2 fused to GST (Wilson et al., 2012). This procedure led to three samples: “GST-FT” represents the total Rpo21, “MD” represents the ubiquitinated Rpo21, “GST” represent the non-specific binding to a GST column. Rpo21-K1246-HTP was analysed using a standard CRAC procedure. All protein genes were tagged at their endogenous locus and were expressed under the control of their endogenous promoter. We have included duplicates samples and an untagged BY4741 negative control.