Quelling targets the rDNA locus and functions in rDNA copy number control

<p>Abstract</p> <p>Background</p> <p>RNA silencing occurs in a broad range of organisms. Although its ancestral function is probably related to the genome defense mechanism against repetitive selfish elements, it has been found that RNA silencing regulates different cellular processes such as gene expression and chromosomal segregation. In <it>Neurospora crassa</it>, a RNA silencing mechanism, called quelling, acts to repress the expression of transgenes and transposons, but until now no other cellular functions have been shown to be regulated by this mechanism.</p> <p>Results</p> <p>Here, we detected by northern blotting endogenous short interfering RNA (siRNAs) from the repetitive ribosomal DNA locus (rDNA) that are loaded onto the argonaute protein QDE-2. Moreover, we found a bidirectional transcription that can generate double strand RNA (dsRNA) molecules. Interestingly, quelling mutants have a reduced rDNA gene copy number.</p> <p>Conclusion</p> <p>Our finding could suggest a new biological function for RNA silencing in the maintenance of the integrity and stability of the <it>Neurospora </it>rDNA locus.</p

The ZFP-1(AF10)/DOT-1 Complex Opposes H2B Ubiquitination to Reduce Pol II Transcription

The inhibition of transcriptional elongation plays an important role in gene regulation in metazoans, including C. elegans. Here, we combine genomic and biochemical approaches to dissect a role of ZFP-1, the C. elegans AF10 homolog, in transcriptional control. We show that ZFP-1 and its interacting partner DOT-1.1 have a global role in negatively modulating the level of polymerase II (Pol II) transcription on essential widely expressed genes. Moreover, the ZFP-1/DOT-1.1 complex contributes to progressive Pol II pausing on essential genes during development and to rapid Pol II pausing during stress response. The slowing down of Pol II transcription by ZFP-1/DOT-1.1 is associated with an increase in H3K79 methylation and a decrease in H2B monoubiquitination, which promotes transcription. We propose a model wherein the recruitment of ZFP-1/DOT-1.1 and deposition of H3K79 methylation at highly expressed genes initiates a negative feedback mechanism for the modulation of their expression

A Conserved PHD Finger Protein and Endogenous RNAi Modulate Insulin Signaling in Caenorhabditis elegans

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16–dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.Leukemia & Lymphoma Society of America (3260-07 Special Fellow Award)Arnold and Mabel Beckman Foundation (Young Investigator Award)United States. National Institutes of Health (Director's New Innovator Award (1 DP2 OD006412-01))United States. National Institutes of Health (grant GM66269)modENCODE (grant U01 HG004270)United States. National Institutes of Health (training grant 5T32 GM07088-34

Small RNAs in epigenetic inheritance: from mechanisms to trait transmission

International audienceInherited information is transmitted to progeny primarily by the genome through the gametes. However, in recent years, epigenetic inheritance has been demonstrated in several organisms, including animals. Although it is clear that certain post-translational histone modifications, DNA methylation, and noncoding RNAs regulate epigenetic inheritance, the molecular mechanisms responsible for epigenetic inheritance are incompletely understood. This review focuses on the role of small RNAs in transmitting epigenetic information across generations in animals. Examples of documented cases of transgenerational epigenetic inheritance are discussed, from the silencing of transgenes to the inheritance of complex traits, such as fertility, stress responses, infections, and behavior. Experimental evidence supporting the idea that small RNAs are epigenetic molecules capable of transmitting traits across generations is highlighted, focusing on the mechanisms by which small RNAs achieve such a function. Just as the role of small RNAs in epigenetic processes is redefining the concept of inheritance, so too our understanding of the molecular pathways and mechanisms that govern epigenetic inheritance in animals is radically changing

Global Run-On sequencing to measure nascent transcription in C. elegans

International audienceGlobal Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode Caenorhabditis elegans to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021)