Genome-wide prediction and analysis of yeast RNase III-dependent snoRNA processing signals
ABSTRACT: In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA. RNA from wild type, rnt1d and rnt1ts grown at 26 C and 37 C on rich media
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. CLIP-seq was performed in Flag-SIRT7-293T cells.
Project description:The paired-end next-generation sequencing of all small RNAs of less than 200 nucleotides in length from four different human cell lines (SKOV3ip1, MCF-7, BJ-Tielf, INOF) allowed us to determine the exact sequence(s) and variations of human box C/D snoRNAs (small nucleolar RNAs), revealing processing patterns of this class of molecules. Two distinct groups of box C/D snoRNAs were identified based on the position of their ends with respect to their characteristic boxes and the terminal base pairing potential. Short box C/D snoRNAs start sharply 4 or 5 nucleotides upstream of their box C and end 2 or 3 nucleotides downstream of their box D. In contrast, long box C/D snoRNAs start 5 or 6 nucleotides upstream of their box C and end 4 or 5 nucleotides downstream of their box D, increasing the likelihood of formation of a k-turn between their boxes C and D. Sequencing of SKOV3ip1 cells following the depletions of NOP58, a core box C/D snoRNA-binding protein and of RBFOX2, a splicing factor, shows that the short box C/D snoRNA forms are significantly more affected by the depletion of RBFOX2 while the long snoRNA forms, which display more canonical box C/D snoRNA features, are significantly more affected by the depletion of NOP58. Together the data suggest that box C/D snoRNAs are divided into at least two groups of RNA with distinct maturation and functional preferences. Small RNAs (<200 nucleotides) were isolated from different human cell lines that were either untreated or depleted of NOP58 or RBFOX2 using specific siRNAs. The resulting libraries were multiplexed and paired-end sequenced using Illumina HiSeq.
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:We performed ChIP seq experiment in MDA-MB-134 cell line in order to map the estrogen receptor alpha (ER) binding sites following the estrogen treatment in an ILC model. We have characterized the genome wide recruit of ER and scaned the binding sites for the presence of cofactor motifs. The binding peaks were also correlated to E2 regulated genes in this ILC model. Four samples were subjected to high throughput sequencing: E-ER (estrogen treated followed by ER IP), E-IgG (estrogen treated followed by IgG), V-ER (EtOH treated followed by ER IP) and Input (MCF7 genomic DNA)
Project description: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:Leukemogenesis requires enhanced self-renewal activity, which is induced by specific oncogenes. The underlying molecular mechanisms remain incompletely understood. We transduced mouse lineage negative bone marrow cells (enriched for hematopoietic stem and progenitor cells) with retrovirus expressing leukemic oncogene AML1-ETO9a, MYC and MLL-AF9 as well as empty vector (MIG). We found that all three oncogenes enhanced snoRNA formation. High abundance of snoRNAs was observed in primary human AML specimens with the notable exception of NPM1 mutant AML. Leukemogenesis by AML1-ETO required expression of the groucho related Amino Enhancer of Split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, loss of C/D box snoRNAs with concomitant loss of rRNA 2’-O-methylation resulted in decreased leukemia self-renewal potential.In summary, we identified C/D box snoRNAs and rRNA 2’-O-methylation as critical determinants of leukemic stem cell activity. Overall design: We used small RNA-Seq to determine the expression profile of small nucleolar RNA (snoRNA) in 63 primary AML patient samples. To further investigate the role of AES and DDX21 in snoRNA formation we also analyzed kasumi-1 cell lines with shRNA based downregulation of AES or DDX21. Furthermore, we show that mouse snoRNAs are induced by leukemia oncogene AML-ETO9a, MLL-AF9 and MYC in mouse lineage negative bone marrow cells.
Project description:Background:Breast cancer is the second most frequent type of cancer affecting women. We are increasingly aware that changes in mRNA splicing are associated with various characteristics of cancer. The most deadly aspect of cancer is metastasis, the process by which cancer spreads from the primary tumor to distant organs. However, little is known specifically about the involvement of alternative splicing in the formation of macroscopic metastases. Our study investigates transcript isoform changes that characterize tumors of different abilities to form growing metastases. Results:To identify alternative splicing events (ASEs) that are associated with the fully metastatic phenotype in breast cancer, we used Affymetrix Exon Microarrays to profile mRNA isoform variations genome-wide in weakly metastatic (168FARN and 4T07) and highly metastatic (4T1) mammary carcinomas. Statistical analysis identified significant expression changes in 7606 out of 155,994 (4%) exons and in 1725 out of 189,460 (1%) intronic regions, which affect 2623 out of 16,654 (16%) genes. These changes correspond to putative alternative isoforms - several of which are novel - that are differentially expressed between tumors of varying metastatic phenotypes. Gene pathway analysis showed that 1224 of genes expressing alternative isoforms were involved in cell growth, cell interactions, cell proliferation, cell migration and cell death and have been previously linked to cancers and genetic disorders. We chose ten predicted splice variants for RT-PCR validation, eight of which were successfully confirmed (MED24, MFI2, SRRT, CD44, CLK1 and HNRNPH1). These include three novel intron retentions in CD44, a gene in which isoform variations have been previously associated with the metastasis of several cancers. Conclusions:Our findings reveal that various genes are differently spliced and/or expressed in association with the metastatic phenotype of tumor cells. Identification of metastasis-specific isoforms may contribute to the development of improved breast cancer stage identification and targeted therapies. Keywords: Seek pre-mRNA changes associated with the fully metastatic phenotype in breast cancer We used RNA tumor tissues derived from three murine mammary carcinoma cell lines (168FARN, 4T07 and 4T1); four biological replicates of 168FARN, four biological replicates of 4T07, and four biological replicates of 4T1 were hybridized independently at McGill university site.
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: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:Box C/D-type small nucleolar RNAs (snoRNAs) are functional RNAs responsible for mediating 2’-O-ribose methylation of ribosomal RNAs (rRNAs) within the nucleolus. Previously, in relation to a novel chromosomal translocation in a human B-cell lymphoma, we identified U50HG, a non-protein-coding gene that hosted a box C/D-type U50 snoRNA within its intron. To investigate the physiological importance of the U50 snoRNA and its involvement in tumorigenesis, we generated a mouse model deficient in mouse U50 (mU50) snoRNA expression without altering the expression of mouse mU50 host-gene, mU50HG-b. The established mU50 snoRNA-deficient mice showed a significant reduction of mU50 snoRNA expression and the corresponding target rRNA methylation in various organs. Lifelong phenotypic monitoring showed that the mU50-deficient mice looked almost normal without accelerated tumorigenicity; however, a notable difference was the propensity for anomalies in the lymphoid organs. B-cells were isolated from spleens of DmU50(HG-b) mice or wild-type C57BL/6J with antibody-conjugated magnetic beads system (Myltenyi Biotec). Total RNA was purified with QIAGEN RNeasy Micro kit. Affymetrix GeneChip® Microarrays (Mouse Expression 430 2.0 Array) were used.