Chromatin Compaction by Small RNAs and the Nuclear RNAi Machinery in C. elegans

Brandon D Fields,Scott Kennedy

doi:10.1038/s41598-019-45052-y

Brandon D Fields, Scott Kennedy

Open Access

https://doi.org/10.1038/s41598-019-45052-y

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Abstract

DNA is organized and compacted into higher-order structures in order to fit within nuclei and to facilitate gene regulation. Mechanisms by which higher order chromatin structures are established and maintained are poorly understood. In C. elegans, nuclear-localized small RNAs engage the nuclear RNAi machinery to regulate gene expression and direct the post-translational modification of histone proteins. Here we confirm a recent report suggesting that nuclear small RNAs initiate or maintain chromatin compaction states in C. elegans germ cells. Additionally, we show that experimentally provided small RNAs are sufficient to direct chromatin compaction and that this compaction requires the small RNA-binding Argonaute NRDE-3, the pre-mRNA associated factor NRDE-2, and the HP1-like protein HPL-2. Our results show that small RNAs, acting via the nuclear RNAi machinery and an HP1-like protein, are capable of driving chromatin compaction in C. elegans.

Highlights

RNAi is an evolutionarily conserved gene regulatory mechanism triggered by double-stranded RNA. dsRNA is recognized and processed by Dicer-like enzymes into small interfering RNAs of 21–25 nucleotides in length17. siRNAs are bound by Argonaute (AGO) proteins to form ribonucleoprotein complexes that use the sequence information contained within siRNAs to regulate complementary RNAs in trans[18]
In C. elegans, the GHKL ATPase MORC-1, a component of the nuclear RNAi machinery, promotes chromatin organization and chromatin compaction in adult C. elegans germ cells, suggesting that siRNAs, acting via the nuclear RNAi machinery and MORC-1, regulate chromatin compaction in C. elegans[40]. To further test this idea, we asked if two other components of the C. elegans nuclear RNAi machinery: the germline expressed nuclear RNAi AGO HRDE-1, and the nuclear RNAi factor NRDE-2, were, like MORC-1, needed for chromatin organization and compaction in adult C. elegans germ cells
The analysis indicated that wild-type, nrde-2(−), and hrde-1(−) nuclei stained with similar amounts of DAPI, suggesting that the increased in space occupied by chromatin in hrde-1(−) or nrde-2(−) nuclei is due to chromatin decompaction and not increased DNA content (Fig. S2)

Summary

Introduction

RNAi is an evolutionarily conserved gene regulatory mechanism triggered by double-stranded RNA (dsRNA). dsRNA is recognized and processed by Dicer-like enzymes into small interfering RNAs (siRNAs) of 21–25 nucleotides in length17. siRNAs are bound by Argonaute (AGO) proteins to form ribonucleoprotein complexes that use the sequence information contained within siRNAs to regulate complementary RNAs in trans (via Watson-Crick base pairing)[18]. SiRNAs are found in nuclei where they bind nascent RNAs to co-transcriptionally regulate gene expression as well as direct the deposition of H3K9me[3] on chromatin (termed nuclear RNAi)[18]. An axis of nuclear small RNAs, H3K9 methytransferases, and HP1-like proteins direct heterochromatin formation and chromatin compaction in many eukaryotes. We show that two additional components of the C. elegans nuclear RNAi machinery are required for normal chromatin compaction and chromatin organization in the germline, supporting the model that endo siRNAs regulate chromatin compaction in nematodes. We show that siRNAs are sufficient to direct chromatin compaction in the soma and that this process requires a nuclear RNAi Ago (NRDE-3) as well as the HP1-like factor HPL-2. Our results support a model in which nuclear- localized small regulatory RNAs are important mediators of chromatin organization and compaction in C. elegans

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