Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

ABSTRACT: We provide evidence of the generation of Rdp1-mediated secondary siRNAs in vivo in fission yeast. Secondly, we show that the presence of Ago1-associated siRNAs does not guarantee robust silencing. We obtained sequences from gel purified small RNAs of wt and rdp1delta fission yeast cells and FLAG-ago1 associated small RNAs.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Alastair Kerr

PROVIDER: E-GEOD-20330 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.

Simmer Femke F Buscaino Alessia A Kos-Braun Isabelle C IC Kagansky Alexander A Boukaba Abdelhalim A Urano Takeshi T Kerr Alastair R W AR Allshire Robin C RC

EMBO reports 20100108 2

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of ...[more]

PMID: 20062003

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Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

Dataset Information

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

Publications

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.

Similar Datasets

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