The post-transcriptional gene silencing machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in Neurospora crassa

Post-transcriptional gene silencing (PTGS) involving small interfering RNA (siRNA)-directed degradation of RNA transcripts and transcriptional silencing via DNA methylation have each been proposed as mechanisms of genome defence against invading nucleic acids, such as transposons and viruses. Furthermore, recent data from plants indicates that many transposons are silenced via a combination of the two mechanisms, and siRNAs can direct methylation of transposon sequences. We investigated the contribution of DNA methylation and the PTGS pathway to transposon control in the filamentous fungus Neurospora crassa. We found that repression of the LINE1-like transposon, Tad, requires the Argonaute protein QDE2 and Dicer, each of which are required for transgene-induced PTGS (quelling) in N.crassa. Interestingly, unlike quelling, the RNA-dependent RNA polymerase QDE1 and the RecQ DNA helicase QDE3 were not required for Tad control, suggesting the existence of specialized silencing pathways for diverse kinds of repetitive elements. In contrast, Tad elements were not significantly methylated and the DIM2 DNA methyltransferase, responsible for all known DNA methylation in Neurospora, had no effect on Tad control. Thus, an RNAi-related transposon silencing mechanism operates during the vegetative phase of N.crassa that is independent of DNA methylation, highlighting a major difference between this organism and other methylation-proficient species

Comprehensive RNA dataset of AGO2 associated RNAs in Jurkat cells following miR-21 over-expression

AbstractWe set out to identify miR-21 targets in Jurkat cells using a high-throughput biochemical approach (10.1016/j.biochi.2014.09.021 [1]). Using a specific monoclonal antibody raised against AGO2, RISC complexes were immunopurified in Jurkat cells over-expressing miR-21 following lentiviral trasduction as well as in Jurkat control cells lines. A parallel immunoprecipitation using isotype-matched rat IgG was performed as a control. AGO2 associated mRNAs were profiled by microarray (GEO: GSE37212). AGO2 bound miRNAs were profiled by RNA-seq

Efficient gene silencing by expression of double stranded RNA in Neurospora crassa

In Neurospora crassa, sequence-specific inhibition of endogenous genes can be induced by the introduction of transgenic DNA homologous to the target gene, through the mechanism of post-transcriptional gene silencing (PTGS) known as quelling. The application of this strategy to inactivate genes in N. crassa has, to date, been restricted by a limited silencing efficiency and instability of the silenced phenotype. In this study we show that the use of constructs that express hairpin double stranded RNA (dsRNA) permits efficient gene silencing by-passing limiting events in the quelling triggering process occurring upstream of dsRNA production. We found that silenced strains expressing a hairpin RNA displayed higher phenotypic stability compared with quelled strains. Moreover, we show that gene silencing can be modulated by expressing the double stranded RNA from an inducible promoter. Together these results make this method suitable for producing hypomorphic mutants in N. crassa. © 2004 Elsevier Inc. All rights reserved

Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora

Small RNA molecules have been found to be specifically associated with posttranscriptional gene silencing (PTGS) in both plants and animals. Here, we find that small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 genes previously shown to be essential for gene silencing, but does not depend on a functional qde-2, indicating that this gene is involved in a downstream step of the gene silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation

Microarray dataset of Jurkat cells following miR-93 over-expression

The dataset presented here represents a microarray experiment of Jurkat cell line over-expressing miR-93 after lentiviral transgenic construct transduction. Three biological replicates have been performed.We further provide normalized and processed data, log2 Fold Change based ranked list and GOterms resulting table. The raw microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number ArrayExpress: E-MTAB-4588. Keywords: miR-93, T-lymphocytes, Homo sapiens, Microarra

Bisulfite miRNA-seq reveals widespread CpG and non-CpG 5-(hydroxy)methyl-Cytosine in human microRNAs

In the last decade, the field of epitranscriptomics highlighted a wide array of post-transcriptional modifications in human RNAs, including microRNAs (miRNAs). Recent reports showed that human miRNAs undergo cytosine methylation. We describe the first high-throughput NGS based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Our method uncovers that miRNAs undergo widespread cytosine modification in various sequence contexts.Furthermore, validation of our data with specific antibodies reveals both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement towards the understanding of the functional implications of this modification in miRNAs. MAmBA is available for download at https://github.com/flcvlr/MAmBA