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A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

ABSTRACT: Long non-coding RNAs (lncRNAs) regulate diverse biological pathways. Unlike protein coding genes, where methods to comprehensibly study their functional roles in cellular systems are available, techniques to systematically investigate lncRNAs have largely remained unexplored. Here, we report a technology for combined Knockdown and Localization Analysis of Non-coding RNAs (c-KLAN) that merges phenotypic characterization and localization approaches to study lncRNAs. Using a library of endoribonuclease prepared short interfering RNAs (esiRNAs) coupled with a pipeline for synthesizing labeled riboprobes for RNA fluorescence in situ hybridization (FISH), we demonstrate the utility of c-KLAN by identifying a novel transcript Panct1 (Pluripotency associated non-coding transcript 1) that regulates embryonic stem cell identity. We postulate that c-KLAN should be generally useful in the discovery of lncRNAs implicated in various biological processes.

ORGANISM(S): Mus musculus

PROVIDER: GSE29798 | GEO | 2012/02/15

SECONDARY ACCESSION(S): PRJNA141015

REPOSITORIES: GEO

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A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

Project description:Long non-coding RNAs (lncRNAs) regulate diverse biological pathways. Unlike protein coding genes, where methods to comprehensibly study their functional roles in cellular systems are available, techniques to systematically investigate lncRNAs have largely remained unexplored. Here, we report a technology for combined Knockdown and Localization Analysis of Non-coding RNAs (c-KLAN) that merges phenotypic characterization and localization approaches to study lncRNAs. Using a library of endoribonuclease prepared short interfering RNAs (esiRNAs) coupled with a pipeline for synthesizing labeled riboprobes for RNA fluorescence in situ hybridization (FISH), we demonstrate the utility of c-KLAN by identifying a novel transcript Panct1 (Pluripotency associated non-coding transcript 1) that regulates embryonic stem cell identity. We postulate that c-KLAN should be generally useful in the discovery of lncRNAs implicated in various biological processes. In this experiment, the levels of Panct1(AK156552) have been depleted by RNAi and the expression levels of all genes have been monitored 72 hours post transfection with esiRNAs against Panct1. An esiRNA against non-targeting Luciferase was used as a negative control.

2012-02-15 | E-GEOD-29798 | biostudies-arrayexpress

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A Hidden Human Proteome Encoded by the “Non-Coding” Genes

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Sirena1: The most abundant lncRNA in mouse oocytes brings together post-transcriptional regulations and mitochondrial distribution (rat wild-type GV oocytes)

Project description:Long non-coding RNAs (lncRNAs) are a large group of rapidly evolving non-coding RNAs. Tens of thousands of lncRNAs were identified but function has been assigned to relatively few. LncRNAs expressed in mouse oocytes have been annotated but their contribution to oocyte development and early development remains unknown. Here, we report evolutionary history and functional analysis of Sirena1, maternal lncRNA that is the most expressed maternal lncRNA and 10th most abundant Pol II transcript in mouse oocytes.

2019-09-30 | GSE137562 | GEO

Sirena1: The most abundant lncRNA in mouse oocytes brings together post-transcriptional regulations and mitochondrial distribution (mouse dev_stages)

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GRID-seq studies of small intestinal epithelial cells from wildtype mice

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Genome-wide resources of endoribonucleotide-prepared short interfering RNAs for specific loss-of-function studies

Project description:RNA interference (RNAi) has become an important technique for loss-of-gene functionstudies in mammalian cells. To achieve reliable results in an RNAi experiment, efficientand specific silencing triggers are required. Here we present genome-wide data sets forthe production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. The database RiDDLE was installed for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective, and specific alternative to currently available mammalian RNAi resources. Consensus gene lists used to generate the clusters appearing in the associated publication have been linked as supplementary files at the foot of the Series record. There is a consensus genelist for each gene in the study: GSE6807_KIAA1387_ConsensusGeneList.txt GSE6807_MAPK14_ConsensusGeneList.txt Keywords: RNAi, off-target effects, esiRNA

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