Project description:We combined cryosectining of oocytes along the animal-vegetal axis (first developmental axis) and RNA-Seq to determine localization profiles of coding and noncoding RNAs. It provides complete view on RNA localization. We found that nearly all RNAs are localized, but only small percentage is actively transported during oogenesis.

2018-05-31 | GSE104848 | GEO

High-throughput identification of RNA nuclear localization signals

Project description:Since the sequencing of the human genome, one of the biggest surprises has been the annotation of thousands of long noncoding RNAs (lncRNAs). Although lncRNAs and mRNAs are similar in many ways, it has become clear they differ in localization properties with lncRNAs being more nuclear enriched and in several cases exclusively nuclear. Yet the RNA based sequences that determine nuclear localization remain poorly understood. Towards the goal of systematically dissecting the lncRNA sequences that impart nuclear enrichment, we developed a massively parallel reporter assay (MPRA). Unlike previous MPRAs that determine motifs important for transcriptional regulation, we have modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 lncRNAs. Using this approach, we identified 109 distinct, conserved nuclear enrichment regions, originating from 29 distinct lncRNAs. We also discovered two shorter motifs that are enriched in our nuclear enrichment regions—one specific to XIST, and a more general motif found in 21 different lncRNAs. We further validated the autonomous functionality of the XIST motif and three nuclear enrichment regions by single molecule RNA FISH. Taken together, these results give the first glimpse of sequence elements responsible for the nuclear enrichment of this critical class of RNA molecules.

2018-01-10 | GSE98828 | GEO

Pontin functions as an essential coactivator for Oct4 target genes and lincRNAs in embryonic stem cells.

Project description:The actions of transcription factors, chromatin modifiers, and noncoding RNAs are crucial for the programming of cellular states. Although chromatin remodeling factors regulate the functional status of cells including pluripotency and differentiation, how they cross-talk with embryonic stem (ES) cell-specific transcription factors and noncoding RNAs to coordinate networks controlling of ES cell identity remain unknown. Here, we find that Pontin chromatin remodeling factor plays an essential role as a coactivator for Oct4 target genes and large intergenic noncoding RNAs (lincRNAs) in ES cells. mRNA- and ChIP-sequencing analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in maintenance of ES cells. Intriguingly, Oct4-dependent coactivator function of Pontin extends to transcription of lincRNAs that are mainly involved in repression of differentiation in ES cells. Together, our findings demonstrate newly identified Oct4-Pontin-lincRNA module plays critical roles in the ES cell circuitry to orchestrate cell fate determination program.

2015-04-08 | GSE58206 | GEO

Antiviral RNA interference in mammalian cells

Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals.

2013-10-10 | GSE43153 | GEO

Antiviral RNA interference in mammalian cells

2013-10-10 | E-GEOD-43153 | biostudies-arrayexpress

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

Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes

Project description:Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes

| PRJEB5049 | ENA