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The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

Piwi-interacting RNAs (piRNAs) are RNA effectors with key roles in maintaining genome integrity and promoting fertility in metazoans. In Caenorhabditis elegans loss of piRNAs leads to a transgenerational sterility phenotype. The plethora of piRNAs and their ability to silence transcripts with imperfect complementarity have raised several (non-exclusive) models for the underlying drivers of sterility. Here, we report the extranuclear and transferable nature of the sterility driver, its suppression via mutations disrupting the endogenous RNAi and poly-uridylation machinery, and copy-number amplification at the ribosomal DNA locus. In piRNA-deficient animals, several small interfering RNA (siRNA) populations become increasingly overabundant in the generations preceding loss of germline function, including ribosomal siRNAs (risiRNAs). A concomitant increase in uridylated sense rRNA fragments suggests that poly-uridylation may potentiate RNAi-mediated gene silencing of rRNAs. We conclude that loss of the piRNA machinery allows for unchecked amplification of siRNA populations, originating from abundant highly structured RNAs, to deleterious levels.

The germline genome is guarded against invading foreign genetic elements by small RNA-dependent gene-silencing pathways. Components of these pathways localize to, or form distinct aggregates in the vicinity of, germ granules. These components and their dynamics in and out of granules are currently being intensively studied. Here, we report the identification of PLP-1, a Caenorhabditis elegans protein related to the human single-stranded nucleic acid-binding protein Pur-alpha, as a component of germ granules in C. elegans. We show that PLP-1 is essential for silencing different types of transgenes in the germ line and for suppressing the expression of several endogenous genes controlled by the germline gene-silencing pathways. Our results reveal that PLP-1 functions downstream of small RNA biogenesis during initiation of gene silencing. Based on these results and the earlier findings that Pur-alpha proteins interact with both RNA and protein, we propose that PLP-1 couples certain RNAs with their protein partners in the silencing complex. PLP-1 orthologs localized on RNA granules may similarly contribute to germline gene silencing in other organisms.

Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest., In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the “virome” keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265–269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270–273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences. IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic datasets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic dataset can be readily queried using web-based tools, meta-searches through all such datasets are less accessible. In this brief communication, we demonstrate such a meta-meta-genomic approach, examining close matches to the Wuhan coronavirus 2019-nCoV in all high-throughput sequencing datasets in the NCBI Sequence Read Archive accessible with the keyword “virome”. In addition to the homology to bat coronaviruses observed in descriptions of the 2019-nCoV sequence (F. Wu et al. 2020, Nature, doi.org/10.1038/s41586-020-2008-3; P. Zhou et al. 2020, Nature, doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in a metavirome dataset generated from the lungs of deceased Pangolins reported by Liu et al. (Viruses 11:11, 2019, http://doi.org/10.3390/v11110979). Our observations are relevant to discussions of the derivation of 2019-nCoV and illustrate the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences.ImportanceMeta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

The germline genome is guarded against invading foreign genetic elements by small RNA-dependent gene-silencing pathways. Components of these pathways localize to, or form distinct aggregates in the vicinity of, germ granules. These components and their dynamics in and out of granules are currently being intensively studied. Here, we report the identification of PLP-1, a Caenorhabditiselegans protein related to the human single-stranded nucleic acid-binding protein Pur-alpha, as a component of germ granules in C. elegans. We show that PLP-1 is essential for silencing different types of transgenes in the germ line and for suppressing the expression of several endogenous genes controlled by the germline gene-silencing pathways. Our results reveal that PLP-1 functions downstream of small RNA biogenesis during initiation of gene silencing. Based on these results and the earlier findings that Pur-alpha proteins interact with both RNA and protein, we propose that PLP-1 couples certain RNAs with their protein partners in the silencing complex. PLP-1 orthologs localized on RNA granules may similarly contribute to germline gene silencing in other organisms.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.

R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae. Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA:DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3Δ, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA:DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNase H act as guardians of the genome.

Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo. DOI: http://dx.doi.org/10.7554/eLife.00505.001, eLife digest Cells with an unusually large number of mutations—either in the form of changes to the DNA sequence or changes in the number or structure of chromosomes—are said to show genome instability. Although these mutations sometimes boost an organism's chances of survival and reproduction, they more often have detrimental effects, which can include cancer. Genome instability can arise as a result of mistakes occurring during the repair of damaged DNA, or due to inappropriate hybridization of RNA to its DNA template. These RNA–DNA hybrids had been thought to occur strictly during the transcription of DNA into RNA. During this process, the two strands of the DNA molecule separate behind the moving RNA polymerase, and this provides an opportunity for the newly formed RNA to hybridize back to its DNA template. When these RNA–DNA hybrids persist, they give rise to DNA damage that leads to genome instability. Although much is known about the factors that prevent the formation of hybrids, or promote their removal, little is known about how hybrids form in the first place. Now, Wahba et al. have identified one such mechanism in the model yeast, Saccharomyces cerevisiae. It involves a protein called Rad51p, which helps to join stretches of nucleic acids together to repair breaks in DNA. However, Wahba et al. showed that if Rad51p is not properly regulated, it can also trigger the formation of RNA–DNA hybrids; yeast cells that lack the gene for Rad51p showed significantly reduced levels of hybrid formation. Moreover, dysfunctional Rad51p causes RNA sequences to anneal to DNA throughout the genome, rather than just at the site in which the RNA was originally produced. This means that RNA sequences produced during transcription are much more of a threat to genomic stability than previously thought. The work of Wahba et al. presents a paradox in which a protein that is normally involved in repairing DNA can itself cause damage if it is not carefully regulated. It also raises the possibility that the elevated levels of Rad51p expression observed in cancer cells could be a cause, rather than a consequence, of mutations. DOI: http://dx.doi.org/10.7554/eLife.00505.002

Roles for long noncoding RNAs (lncRNAs) in gene expression are emerging, but regulation of the lncRNA itself is poorly understood. We have identified a homeodomain protein, AtNDX, that regulates COOLAIR, a set of antisense transcripts originating from the 3’ end of Arabidopsis FLOWERING LOCUS C (FLC). AtNDX associates with single-stranded DNA rather than double-stranded DNA non–sequence-specifically in vitro, and localizes to a heterochromatic region in the COOLAIR promoter in vivo. Single-stranded DNA was detected in vivo as part of an RNA-DNA hybrid, or R-loop, that covers the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR transcription, which in turn modifies FLC expression. Differential stabilization of R-loops could be a general mechanism influencing gene expression in many organisms.