Project description:Various small RNA libraries from purified microtubules or Xiwi immunoprecipitates or total extract from X.tropicalis or X.laevis egg extract. Various small RNA libraries from single X.tropicalis eggs. Analysis of this series of files is described in the manuscript "Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi".
Project description:Various small RNA libraries from purified microtubules or Xiwi immunoprecipitates or total extract from X.tropicalis or X.laevis egg extract. Various small RNA libraries from single X.tropicalis eggs. Analysis of this series of files is described in the manuscript "Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi". Small RNAs were ligated with linkers and converted to cDNA by reverse transcription. cDNA library was amplified by PCR and was sequenced with either the 454 Genome Sequencer FLX platform or the Illumina GA-II platform.
Project description:Small noncoding (snc) RNAs represent a growing family of transcripts that regulate key cellular processes, including mRNA degradation, translational repression and transcriptional gene silencing. Among these, the PIWI-interacting RNAs (piRNAs), a major class of sncRNAs initially identified in the germline of a variety of species, are now being found to be functionally active also in somatic cells. However, whether the Piwi/piRNA pathway is associated with fundamental biological processes, such as cell cycle progression, remains elusive. Here we investigated the possibility that piRNAs are expressed in liver and modulated during regenerative proliferation of this organ. To this aim, smallRNA-Seq was applied to identify and quantitate expression of these RNAs in rat liver before, during and after the wave of cell proliferation that follows partial hepatectomy (PH). Q-PCR analysis revealed the presence in rat liver of two PIWI (PIWI-Like) subfamily members (PIWIL2/HILI and, to a much lower level, PIWIL4/HIWI2) and other components of the piRNA biogenesis pathways, suggesting that this is present and functional in hepatocytes. Indeed, ~1400 piRNAs originally identified in rat and other mammalian germline cells are expressed in adult rat liver, including 72 that show timed changes in expression during cell cycle progression. Most piRNAs are up-regulated 24-48h after hepatectomy, a timing that corresponds to cell transition through the S phase, and return to basal levels by 168 h, when organ regeneration is completed and hepatocytes reach quiescence. These results indicate that the piRNA pathway is active in somatic cells and, more important, that it is subject to regulation during physiological processes, such as cell proliferation, when piRNAs may exert their regulatory functions on the cell genome and transcriptome. smallRNA-Seq was applied to identify and quantitate expression of RNAs in rat liver before and after partial hepatectomy (PH).
Project description:This study examines the population of transcripts associated with the Xenopus Piwi proteins, Xiwi and Xili, from X.laevis and X.tropicalis. RIP-seq, CLIP-seq, piRNA-seq, and mRNA-seq datasets were integrated to determine how the Xenopus Piwi proteins where using piRNAs and binding interactions to associate with transcripts in gonadal cells.
Project description:Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for germ cell development, but analysis of the molecular mechanisms of these ribonucleoproteins remains challenging in most animal germ cells. To address this challenge, we systematically characterized Xiwi, a Xenopus Piwi homologue, and piRNAs from Xenopus eggs and oocytes. We used the large size of Xenopus eggs to analyze small RNAs at the single cell level, and find abundant piRNAs and large piRNA clusters in the Xenopus tropicalis genome, some of which resemble the Drosophila piRNA-generating flamenco locus. Although most piRNA clusters are expressed simultaneously in an egg, individual frogs show distinct profiles of cluster expression. Xiwi is associated with microtubules and the meiotic spindle, and is localized to the germ plasm--a cytoplasmic determinant of germ cell formation. Xiwi associates with translational regulators in an RNA-dependent manner, but Xenopus tudor interacts with Xiwi independently of RNA. Our study adds insight to piRNA transcription regulation by showing that individual animals can have differential piRNA expression profiles. We suggest that in addition to regulating transposable elements, Xiwi may function in specifying RNA localization in vertebrate oocytes.
Project description:PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that partner with PIWI proteins to protect germline tissues from destabilizing transposon activity. While the aberrant expression of PIWI proteins has been linked with poor outcomes for many cancers, less is known about the expression or function of piRNAs in cancer. We performed array-based piRNA expression profiling in seven pairs of normal and glioblastoma multiforme (GBM) tissue specimens and identified expression of ~350 piRNAs in both tissues and a subset with dysregulated expression in GBM. Several down-regulated piRNAs inhibited proliferation when transfected into glioma cell lines while those equivalently expressed in tumor and normal tissues did not, consistent with piRNA-specific tumor-suppressive roles. Upregulation of the most underexpressed piRNA, piR-8041, was found to induce cell cycle arrest and apoptosis and to alter transcriptional levels of several genes involved in stress and cell survival pathways. Additionally, the volume of intracranial mouse xenograft tumors was significantly reduced for approximately ten days after pre-implantation transfection with piR-8041. Taken together, our study reveals a previously unidentified functional role for piRNAs as tumor suppressors in gliomagenesis, and suggests that restoration of piRNA levels may be a potential strategy for GBM therapy.
Project description:There have been substantial researches focused on the roles of microRNAs (miRNA) and Piwi-interacting RNAs (piRNA) delivered from mammalian spermatozoa, comparatively little is known about the role of sperm-delivered miRNAs and piRNAs within cockerels’ (breeding cock) sperm. A small RNA library of cockerels’ sperm was constructed using Illumine high-throughput sequencing technology.
Project description:Here we report on genome-wide small RNA and transcriptome profiling of blastula, gastrula and neurula-stage Xenopus tropicalis embryos using deep sequencing. By combining bioinformatic analysis of the small RNA-seq dataset with experimental validation, we identify novel microRNAs and Piwi-interacting RNAs. We show that microRNA expression is highly dynamic and spatially localized in early embryos in contrast to Piwi-interacting RNAs, and we validate a novel microRNA prediction pipeline. We go on to identify a new class of small intronic non-coding RNAs, that we name sincRNAs. On a genome-wide scale we find that genes containing sincRNA clusters are transcriptionally repressed. Our data suggest a novel mechanism of sincRNA-mediated gene silencing in vertebrates. Examination of small RNAs and mRNA at 3 stages of Xenopus embryonic development.