ABSTRACT: Small nucleolar RNAs (snoRNAs) dysfunction have been associated with cancer development. We investigated the function of an orphan C/D box class snoRNA, SNORD126, in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cells We used microarrays to identify targets with roles in SNORD126âs activity in Huh-7 cells SNORD126- or EGFP-overexpressed Huh-7 cells were collected and followed by RNA extraction, then hybridized with Affymetrix microarrays. We sought to obtain the differentially expressed genes between the two groups.
Project description:Small nucleolar RNAs (snoRNAs) dysfunction have been associated with cancer development. We investigated the function of an orphan C/D box class snoRNA, SNORD126, in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cells We used microarrays to identify targets with roles in SNORD126’s activity in Huh-7 cells
Project description:H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.
Project description:H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.
Project description:H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.
Project description:Background: Small nucleolar RNAs (snoRNAs) are mid-size non-coding RNAs required for ribosomal RNA modification, implying a ubiquitous tissue distribution linked to ribosome synthesis. However, increasing numbers of studies identify extra-ribosomal roles of snoRNAs in modulating gene expression, suggesting more complex snoRNA abundance patterns. Therefore, there is a great need for mapping the snoRNome in different human tissues as the blueprint for snoRNA functions. Results: We used a low structure bias RNA-Seq approach to accurately quantify snoRNAs and compare them to the entire transcriptome in seven healthy human tissues (breast, ovary, prostate, testis, skeletal muscle, liver and brain). We identify 475 expressed snoRNAs categorized in two abundance classes that differ significantly in their function, conservation level and correlation with their host gene: 390 snoRNAs are uniformly expressed and 85 are enriched in the brain or reproductive tissues. Most tissue-enriched snoRNAs are embedded in lncRNAs and display strong correlation of abundance with them, whereas uniformly expressed snoRNAs are mostly embedded in protein-coding host genes and are mainly non- or anticorrelated with them. 59% of the non-correlated or anticorrelated protein-coding host gene/snoRNA pairs feature dual-initiation promoters, compared to only 16% of the correlated non-coding host gene/snoRNA pairs. Conclusions: Our results demonstrate that snoRNAs are not a single homogeneous group of housekeeping genes but include highly regulated tissue-enriched RNAs. Indeed, our work indicates that the architecture of snoRNA host genes varies to uncouple the host and snoRNA expressions in order to meet the different snoRNA abundance levels and functional needs of human tissues.
Project description:Small nucleolar RNAs (snoRNA) are non-coding RNAs known for guiding RNA modifications including 2ʹ-O-methylation (Nm) and pseudouridine (Ψ). While snoRNAs may also interact with other RNAs such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome-wide. Here we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues. Many interactions occur outside of snoRNA-guided rRNA modification sites, hinting at non-canonical functions beyond RNA modification. We find that one of these snoRNAs, SNORA73, targets mRNAs that encode secretory proteins and membrane proteins. SNORA73 also interacts with 7SL RNA, part of the signal recognition particle (SRP) required for protein secretion. The mRNA-SNORA73-7SL RNA interactions enhance the association of the SNORA73-target mRNAs with SRP, thereby facilitating secretion of the encoded proteins.
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:We evaluated the profile of miRNA and snoRNA expression in 7 solitary CRC and matched normal colorectal tissues using the Affymetrix GeneChip miRNA 1.0 array. We found that global dysregulated miRNAs and snoRNAs between cancer tissue and normal mucosa in solitary CRC. Our findings firstly implicates that dysregulation of snoRNAs and miRNA may play important role in the cancinogenesis and present therapeutic targets for solitary CRC. Examination of microRNA and snoRNA expression in cancer and matched normal tissues of solitary CRC
Project description:Small nucleolar RNA (snoRNA) are non-coding RNAs, which participate in the chemical modification of ribosomal RNAs (rRNAs) and small nuclear RNAs. However, the roles of snoRNA in homeostasis of hematopoietic stem cells(HSCs) have not been studied. We isolated four hematopoietic stem and progenitor cells (HSPCs) (LT-HSCs, IT-HSCs, ST-HSCs, and MPPs) from the bone marrow (BM) of C57BL/6 mice and performed small RNA-seq. We found SnoRNAs belonging to SNORD113-114 cluster were specifically enriched in LT-HSCs, and their expression decreased dramatically with HSC differentiation. To explore function of snoRNAs in SNORD113-114 cluster in HSCs, we established maternally KO mice by CRISPR-Cas9 technology. Here, we used a modified small RNA-seq method to examine snoRNA profile in KO mice.
Project description:Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2’-O-ribose methylation through base pairing with targeted sequences. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA. However, these studies provide a narrow portrait of snoRNA targets forming under specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA machine learning interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, and position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. The interactions of snoRNAs are not randomly distributed but often accumulate in functionally related transcripts sharing common regulatory elements suggesting coordinated regulatory function. The wide scope of snoGloBe makes it an excellent tool for discovering viral RNA targets, which is evident from its capacity to identify snoRNAs targeting SARS-CoV-2 RNA, known to be heavily methylated. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.