MORC1 represses transposable elements in the mouse male germ line
ABSTRACT: The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA methylated and silent genes. MORC1 deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homolog DNMT3L. Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation. This data includes: 47 RNA-seq, 4 smRNA-seq, 6 BS-seq, and 2 ChIP-seq datasets
Project description:In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use piRNA guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2-deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 suggesting that the proteins have independent roles in establishing DNA methylation patterns. Small RNA profiles (19-30 nt range) of embryonic (E16.5) and post-natal (P10) testis of Miwi2 mutant mice and matched heterozygote controls. mRNA profiles of embryonic testis (E16.5) of heterozygote control mice and of postnatal testis (P10) of Miwi2 and Mili mutants and heterozygote controls. CpG methylation BS-seq profile of postnatal (P10) spermatocytes of Miwi2 mutant mice and matched heterozygote controls.
Project description:Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci. For example, only one C. elegans transposon is a known piRNA target. Here we show that, in mutants lacking the Piwi Argonaute PRG-1 and associated piRNAs (21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression and depletion of an amplified RNAdependent RNA polymerase (RdRP)-derived species of small RNA termed 22G-RNAs. Sequences depleted of 22G-RNAs are enriched nearby potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing. Examine small RNA population changes in prg-1 and rescued strains
Project description:In this study, we analyzed function of the rice RDR701 and various DCLs proteins in small RNA processing. small RNA deep sequencing analyses revealed that RDR701 and OsDCL4 is involved in tasiRNAs and 21- and 24-nt phasiRNAs biogenesis. Further analyses indicate RDR701 coupling with OsDCL4 and OsDCL3b are required for the biogenesis of 21- and 24-nt phasiRNAs, respectively. The triggering miRNAs for intial both size phasiRNAs,are also shown RDR701- and OsDCL4-dependent, suggesting two sequential round activity of RDR701 in associated with different DCLs are essential for rice phasiRNAs biogenesis. Four small RNA libraries were constructed from panicles of zhonghua11 (a wild-type Japonica rice variety) and rdr701-1 at high temperature (Day average temperature 32℃) and low temperature (Day average temperature 28℃).
Project description:The osdcl4-1 mutant exhibits much severer developmental defects than dcl4 in Arabidopsis, suggesting that Os DCL4 may process broader substrates in rice. By deep sequencing of small RNAs from different tissues of wild types and osdcl4-1, we revealed that 21-nucleotide siRNAs were largely dependent on Os DCL4. Besides several tasiRNA loci reported in Arabidopsis and rice, over one thousand 21-nucleotide and several dozen 24-nucleotide phased siRNA (phasiRNA) clusters were identified in panicles but not in seedlings and grains. Further analyses identified two conserved 22-nucleotide motifs among the cleavage sites of the 21- and 24- phasiRNA loci, and the cleavage sites of over 90% of 21- and 24-nucleotide phasing clusters were confirmed by PARE/degradome analysis from 93-11 panicles. MiR2118 and miR2275, expressed specifically in panicles, were predicted to trigger cleavages at 21- and 24-nucleotide phasiRNA clusters, respectively. The triggers of phasiRNAs are more dependent on Os DCL4 than Os DCL1. Furthermore, the processing of 21-nucleotide phasiRNAs was largely Os DCL4-dependent, whereas the processing of 24-nucleotide phasiRNAs was slightly affected by Os DCL4, but not by Os DCL3a and Os DCL1. Our results revealed distinct roles of Os DCL4 in a novel 21- and 24-nucleotide phasiRNA biogenesis pathway in rice. Six small RNA libraries were constructed from seedlings and panicles of 93-11 (a wild-type Indica rice variety) and osdcl4-1, as well as those from Nipponbare panicles and seedlings (a wild-type Japonica rice variety).
Project description:Small interfering RNAs (siRNAs) are known to be involved in both transposon silencing and centromere function, leading us to investigate the interplay between these two roles in the Schizosaccharomyces lineage. In S. pombe, the centromeric repeats produce dicer-dependent siRNAs that are required for maintenance of centromeric structure, function and transcriptional silencing via Argonaute-dependent heterochromatin formation13. However, transposons are silenced in S. pombe by RNAi-independent mechanisms and do not produce abundant siRNAs. To investigate whether centromere-directed siRNA production is conserved within the transposon-rich centromeres of S. japonicus, we isolated and sequenced small RNAs from log-phase S. japonicus cultures. The small RNAs have a modal size of 23 nucleotides and 94% map to transposons, both telomeric and centromeric. Isolation and computational analysis of small RNAs from wild-type S. japonicus
Project description:Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. In Drosophila, the reciprocal “Ping-Pong” cycle of piRNA-directed RNA cleavage, catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) through their Slicer activity, is believed to expand the population of antisense piRNAs in response to transposon expression. Whether and how the Slicer activity of AGO3/Aub promotes the process of the secondary piRNA amplification remain unclear. Here we generated transgenic flies that could express AGO3 Slicer mutant forms to ellucidate the Slicer activity of AGO3. small-RNA libraries from 3 samples of D. mel. Ovaries.
Project description:In mammals, piRNA populations are dynamic throughout male germ cell development. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, however, the piRNA population is transposon-poor and restricted to primary piRNAs derived from pachytene piRNA clusters. The mechanism controlling which piRNAs are present at each developmental stage is poorly understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong (secondary amplification) occurs inappropriately in meiotic cells – aberrantly targeting protein-coding genes and lncRNAs. Our data indicate that RNF17 comprises one component of a “refereeing” mechanism that prevents deleterious activity of the meiotic piRNA pathway by ensuring the selective loading of PIWI proteins with products of meiotic piRNA clusters. Examination of small RNA profile in heterozygous and homozygous RNF17 adult testes, pachytene or round spermatid sorted cells
Project description:Transposable elements (TEs) occupy large fraction of metazoan genomes and pose constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small non-coding piwi interacting (pi)RNAs recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. Total of 34 libraries were analyzed. In case of ChIP libraries, every 'input' sample was used for normalization of the respective ChIP (H3K9me3 or H3) sample; the input libraries preceed ChIP libraries in the list below. There are four replicates (input-ChIP pairs) for H3K9me3 ChIP on liver cells, two for ChIP on testicular somatic cells for each genotype (Miwi2 Het and KO); and four for ChIP on male germ cells for each genotype (one from FACS-sorted germ cells, and three from MACS-sorted germ cells). Each replicate has a Het/KO pair, and the respective libraries were cloned from the material isolated from littermates. One H3 ChIP for each genotype is included, with respective inputs.
Project description:In mammals, piRNA populations are dynamic throughout male germ cell development. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, however, the piRNA population is transposon-poor and restricted to primary piRNAs derived from pachytene piRNA clusters1-6. The mechanism controlling which piRNAs are present at each developmental stage is poorly understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong (secondary amplification) occurs inappropriately in meiotic cells – aberrantly targeting protein-coding genes and lncRNAs. Our data indicate that RNF17 comprises one component of a “refereeing” mechanism that prevents deleterious activity of the meiotic piRNA pathway by ensuring the selective loading of PIWI proteins with products of meiotic piRNA clusters. Examination of small RNAs isolated from MIWI and MILI IPs of heterozygous and homozygous RNF17 adult testes
Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.