Project description:The dataset contains small RNAs that associated with HrAgo1 during heterologous expression in E. coli. The goal of the study was to determine what type of small RNAs associate with HrAgo1 and from what RNA transcripts these small RNAs are derived
Project description:In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15-18 nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
Project description:Nuclear Argonaute proteins, guided by their bound small RNAs, orchestrate heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms that, besides recruiting heterochromatin effector proteins, are required for this silencing process are poorly understood. Here, we show that the SFiNX complex, the central silencing mediator downstream of nuclear Piwi-piRNA complexes in Drosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX to form a homodimer. The dynein light chain dLC8, a highly conserved dimerization hub protein, mediates homo-dimerization of SFiNX. Point mutations preventing dLC8-mediated SFiNX dimerization result in transposon de-repression and sterility. dLC8’s function can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive rather than a regulated feature of SFiNX. We propose that nucleic-acid stimulated condensate formation enables co-transcriptional silencing through the retention of the target RNA at chromatin, thereby allowing effector proteins to establish heterochromatin at the target locus.
Project description:This SuperSeries is composed of the following subset Series: GSE39746: Argonaute proteins couple chromatin silencing to alternative splicing (exon array) GSE39748: Argonaute proteins couple chromatin silencing to alternative splicing (RNA IP-Seq) Refer to individual Series
Project description:In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.
Project description:Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. Argonaute proteins mediate gene regulation by small RNAs and thereby contribute to cellular homeostasis during diverse physiological process, such as stem cell maintenance, fertilization, and heart development. Over the last decade, remarkable progress has been made toward understanding Argonaute proteins, small RNAs, and their roles in eukaryotic biology. Here, we review current understanding of Argonaute proteins from a structural prospective and discuss unanswered questions surrounding this fascinating class of enzymes.
Project description:Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline deposits proteins and RNAs into oocytes to support these divisions, which lack many of the quality control mechanisms operating in somatic cells undergoing growth. How the composition of the oocyte maternal load is regulated to ensure its ability to support early embryogenesis is not known. Here we describe a small RNA-Argonaute pathway, operating in the C. elegans germline, that ensures early embryonic divisions by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major embryonic phenotype associated with pathway loss. Tuning of target expression is guided by small RNA density, which must ultimately be related to target sequence. Thus, C. elegans employs a single catalytic Argonaute for small RNA-mediated tuning of the mRNA levels of germline-expressed genes that support early embryogenesis. mRNA profiling of 2 replicates each for 3 genotypes of adult-stage C. elegans worms
Project description:In animals Argonaute small-RNA pathways scan germline transcripts to silence self-replicating genetic elements. Little is known however about how endogenous gene expression is recognized and licensed. Here we show that the presence of introns and by inference the process of mRNA splicing prevents default Argonaute-mediated silencing in the C. elegans germline. Silencing of intronless genes is initiated independently of the piRNA pathway but nevertheless engages multiple components of the downstream amplification and maintenance mechanisms that mediate transgenerational silencing including both nuclear and cytoplasmic members of the worm-specific Argonaute gene family (WAGOs). Small RNAs amplified from intronless mRNAs can trans-silence cognate intron-containing genes. Interestingly, a second small RNA-independent cis-acting mode of silencing also acts on intronless mRNAs. Our findings suggest that cues put in place during mRNA splicing license germline gene expression and provide evidence for a splicing-dependent and dsRNA- and piRNA-independent mechanism that can program Argonaute silencing.