Small-RNA transcriptome sequencing of individual cells
Ontology highlight
ABSTRACT:
OTHER RELATED OMICS DATASETS IN: PRJNA321192
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE81287 | GEO | 2016/10/17
SECONDARY ACCESSION(S): PRJNA321192
REPOSITORIES: GEO
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Project description:Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. We report thousands of Ago2 target sites driven by microRNAs, but also a substantial number of Ago2 ‘drivers’ derived from fragments of other small RNAs such as tRNAs, snoRNAs, rRNAs and others. Target validation of several miRNAs delivered by 3’ Quantseq RNA-Seq.
2023-12-24 | GSE221573 | GEO
Project description:Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. We report thousands of Ago2 target sites driven by microRNAs, but also a substantial number of Ago2 ‘drivers’ derived from fragments of other small RNAs such as tRNAs, snoRNAs, rRNAs and others. Target validation of several miRNAs delivered by 3’ Quantseq RNA-Seq.
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Project description:Background: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequenced long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. Results: Analysis of these data sets revealed that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently catalogued. We further found that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Conclusions: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon. PAR-CLIP profiling for snoRNP core proteins NOP56, NOP58, Fibrillarin, and Dyskerin in HEK293 cells. Small RNA profiling using RNA-seq in HEK293 and HeLa cells, small RNA profiling using IP-seq of Ago2 associated small RNAs.
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Project description:Background: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequenced long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. Results: Analysis of these data sets revealed that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently catalogued. We further found that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Conclusions: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
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Project description:The small non-coding RNAs (sncRNAs) are considered as postranscriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs or rRNAs processing may also play important regulatory roles in spermatogenesis. By next generation sequencing (NGS), we characterized the different sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs) at 13.5 dpc, pubertal spermatogonia cells, and mature spermatozoa. In order to assess the potential transmission of the sncRNAs through the mature spermatozoa during fertilization, the sncRNA population detected in male germ cells was also compared with sncRNAs detected in unfertilized mouse oocytes and zygotes. Combining the data obtained by NGS and microarrays from whole PGC and spermatogonia transcriptome, we defined the potential regulatory roles of specific miRNAs and their validated targets. Similar to miRNAs, both the small RNAs derived from snoRNAs and the piRNAs, could be involved in the postranscriptional regulation of mRNA transcripts during the male germ development. Finally, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. These new classes of piRNAs are not generated by the ping-pong pathway and could be the source of primary piRNAs. mRNA analysis of Primordial Germ Cells (PGCs), Spermatogonia cells (SPG), adult testis (AdT) and Gonad-less (GL) embryos. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data.
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Project description:The small non-coding RNAs (sncRNAs) are considered as postranscriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs or rRNAs processing may also play important regulatory roles in spermatogenesis. By next generation sequencing (NGS), we characterized the different sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs) at 13.5 dpc, pubertal spermatogonia cells, and mature spermatozoa. In order to assess the potential transmission of the sncRNAs through the mature spermatozoa during fertilization, the sncRNA population detected in male germ cells was also compared with sncRNAs detected in unfertilized mouse oocytes and zygotes. Combining the data obtained by NGS and microarrays from whole PGC and spermatogonia transcriptome, we defined the potential regulatory roles of specific miRNAs and their validated targets. Similar to miRNAs, both the small RNAs derived from snoRNAs and the piRNAs, could be involved in the postranscriptional regulation of mRNA transcripts during the male germ development. Finally, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. These new classes of piRNAs are not generated by the ping-pong pathway and could be the source of primary piRNAs.
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