Project description:To study effect of VRK1 deletion on spermatogenesis of the mouse, transciptomic analysis of genes in postnatal 8-day testicular cells of wild type and VRK1-deficient Mus musculus was performed. Gene expression in testes from from wild type and VRK1-deficient mutant Mus musculus, respectively, was measured. Four independent experiments for wild type and mutant, respectively, were performed.
Project description:In animal germline cells, Piwi-interacting RNAs (piRNAs) silence retrotransposons through post-transcriptional and transcriptional mechanisms. However, little is known, especially in mammals, about the functions of piRNAs beyond retrotransposon suppression1-5. In mammalian spermatocytes, piRNAs are known to be abundantly expressed6-10. Here, we show that a subset of coding and noncoding RNAs in mouse spermatocytes is degraded by the piRNA pathway. By analyzing the germline trasnscriptome of mice deficient in piRNA biogenesis, we identify hundreds of mRNAs as direct targets of piRNAs. Remarkably, the 3' untranslated region (UTR) of the mRNAs up-regulated in the piRNA pathway mutants are highly enriched with retrotransposon sequenes, implying that these sequences serve as regulatory elements for piRNA-mediated regulation. Furthermore, deficiencies of piRNAs derived from pseudogenes result in increased mRNA levels of their cognate genes, indicating that pseudogenes regulate their functional cognates via piRNAs. Moreover, we identify a large population of testis-enriched long intergenic noncoding RNAs (lincRNAs), some of which are also degraded by the piRNA pathway. Collectively, our results reveal that the piRNA pathway regulates the expression of both mRNAs and lincRNAs in addition to retrotransposon RNAs during meiosis and the key role of retrotransposons and pseudogenes, two major types of genomic sequences, in this regulation by acting as piRNA sources and/or regulatory elements in target RNAs. Small RNAs in Stambp-ps1 mutant testes were sequenced using Illumina HiSeq.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:Testes were collected from 16-day-old wild-type and Hsf5 knockout mice; small RNA libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform, with adapter trimming, extraction of 18–32 nt reads for piRNA analysis, alignment to miRBase, non-coding RNAs, piRNA clusters, and transposable elements (RepeatMasker, mm39), categorization of aligned 25–32 nt piRNAs, and ping-pong analysis using LINE1 and IAP consensus sequences from Repbase, to determine whether HSF5 deficiency affects piRNA biogenesis and transposon silencing during mouse spermatogenesis by assessing piRNA abundance, length distribution, annotation profile, and ping-pong amplification in Hsf5-/- testes
Project description:Piwi-interacting RNAs are 25-32 nt small RNAs bound to Piwi proteins. To know the steps where factors involved in the piRNA biogenesis (MILI, MIWI2, ZUC (MitoPLD), MVH) work, we sequenced small RNAs from mutant mouse testes and analyzed piRNAs.