Project description:For the evaluation of "WIND: A Workflow for pIRNAs aNd beyonD" performance and the transcriptomic approach on small-RNA identification, and particularly on piRNAs, two mouse Adult Cardiomyocytes (aCMs) were used.
Project description:WIND: A Workflow for pIRNAs aNd beyonD, for the evaluation of the performance in piRNAs high expression and low expression conditions, we used Human Testis RNAs (BioChain Institute Inc, Newark, CA, USA) and COLO 205 cell line RNAs (samples are available on ArrayExpress (E-MTAB-8115: Non_treated_Testis_1 and Non_treated_COLO205_1, Non_treated_COLO205_2, Non_treated_COLO205_3).
Project description:For the evaluation of \\"WIND: A Workflow for pIRNAs aNd beyonD\\" performance and the transcriptomic approach on small-RNA identification, and particularly on piRNAs, a synthetic set of 4 piRNA-like molecules was used. Two non-methylated (SS-22 and SS-28) and two methylated (mSS-22 and mSS-28) of two different lengths (22 nt and 28 nt) were included. Spike-ins were chemically synthesised at Exiqon, the pool of 4 molecules was used at three different concentrations, with a final amount of 0.3 x 10^9 (dil_A), 0.3 x 10^10 (dil_B) and 0.3 x 10^11 (dil_C) molecules/ug of RNA. Library preparation and sequencing were done as previously described in Sellitto et al 2019 (doi: https://doi.org/10.3390/cells8111390)
Project description:Monitor the efficiency of "WIND: A Workflow for pIRNAs aNd beyonD" for the identification of single-stranded (SS) spike-in piRNA-like molecules in smallRNA-seq
Project description:Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce the complex wind force signals downstream to nuclear events and the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components of the wind drag force signaling, two force-regulated phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the TurboID overexpression transgenics as a universal control. This quantitative biotinylproteomic work successfully identified 11 and 71 MKK1- and MKK2 - associated proteins, respectively. A WInd-Related Kinase 1 (WIRK1), previously known as Rapidly Accelerated Fibrosarcoma 36 protein (RAF36), was eventually found to be a common interactor for both MKK1 and MKK2 kinases. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the expression of touch-responsive TCH3 and CML38 genes and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1-1 mutant revealed that the WIRK1 gene is involved in both wind response and gravitropism of Arabidopsis, suggesting that WIRK1 protein may serve as the crosstalk point among multiple signal transduction pathways of both gravitropic and wind responses. It is likely that gravity force signaling may be an integral part of the wind mechano-signaling network in various parts of plant organs.
Project description:Current bioinformatics workflows for PIWI-interacting RNA (piRNA) analysis focus primarily on germline-derived piRNAs and piRNA-clusters. Frequently, they suffer from outdated piRNA databases, questionable quantification methods, and lack of reproducibility. Often, pipelines specific to miRNA analysis are used for the piRNA research
Project description:A developmental series of wind-treated Populus leaf tissue was subjected to array analyses in order to address the issue of age-dependent responsiveness to environmental changes. The following developmental stages were defined for the experiment: Y – “youngest leaf” including the shoot tip = smallest fully enrolled leaf; E – “expanded leaf” = oldest leaf that had not reached full leaf thickness; M – “mature leaf” = 5th leaf below E = has reached full leaf expansion and full leaf thickness; O – “old leaf” = 5th leaf below E. Keywords: transcription profiling Two-condition experiment, control (K) vs. Wind-treated (W) leaves. Biological replicates: 3 control (1-3), wind-exposed (1-3), independently grown and harvested. One swap replicate per array.
Project description:A developmental series of wind-treated Populus leaf tissue was subjected to array analyses in order to address the issue of age-dependent responsiveness to environmental changes. The following developmental stages were defined for the experiment: Y – “youngest leaf” including the shoot tip = smallest fully enrolled leaf; E – “expanded leaf” = oldest leaf that had not reached full leaf thickness; M – “mature leaf” = 5th leaf below E = has reached full leaf expansion and full leaf thickness; O – “old leaf” = 5th leaf below E. Keywords: transcription profiling
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. Late spermatocyte mRNA profiles of Miwi+/- and -/- were analyzed by deep sequencing, in triplicate, using Illumina HiSeq.