Protection from feed-forward amplification in an amplified RNAi mechanism.

The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA-directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in Caenorhabditis elegans, we find that feed-forward amplification is limited by biosynthetic and structural distinctions at the RNA level between (1) triggers that can produce amplification and (2) siRNA products of the amplification reaction.

Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets.

miRNAs are post-transcriptional regulators of gene activity that reduce protein accumulation from target mRNAs. Elucidating precise molecular effects that animal miRNAs have on target transcripts has proven complex, with varied evidence indicating that miRNA regulation may produce different molecular outcomes in different species, systems, and/or physiological conditions. Here we use high-throughput ribosome profiling to analyze detailed translational parameters for five well-studied targets of miRNAs that regulate C.

Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint.

Exogenous double-stranded RNA (dsRNA) has been shown to exert homology-dependent effects at the level of both target mRNA stability and chromatin structure. Using C. elegans undergoing RNAi as an animal model, we have investigated the generality, scope and longevity of dsRNA-targeted chromatin effects and their dependence on components of the RNAi machinery.

Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C. elegans.

MicroRNAs (miRNAs) are regulatory molecules that share both biosynthetic derivation (cleavage from short hairpin precursor RNAs) and functional roles (downregulation of specific mRNAs through targeted degradation and/or translational inhibition). A distinct family of small RNAs, termed siRNAs, have some common characteristics but exhibit distinct modes of biosynthesis and function. In this study, we report procedures for purification of a predominant species of miRNA-containing ribonucleoprotein complexes from Caenorhabditis elegans and demonstrate that this population is distinct from the predominant pool of siRNA-containing ribonucleoprotein complexes.

Distinct populations of primary and secondary effectors during RNAi in C. elegans.

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C.

Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae.

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs.

Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae.

The NDT80 gene of Saccharomyces cerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) and mid-sporulation element (MSE) sites are present in the promoter region of the NDT80 gene; these elements have been shown previously to contribute to the regulation of expression of early and middle sporulation-specific genes, respectively, by mediating repression in growing cells and activation at specific times during sporulation. In this study, we have shown that the overlapping windows of URS1- and MSE-mediated repression and activation are responsible for the distinctive premiddle expression pattern of the NDT80 gene.