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Key Points Functional diversification and expansion of silencing pathways in plants relies on duplication of DICER-LIKE proteins (DCLs) and ARGONAUTE proteins (AGOs). The main small-RNA classes in plants are microRNAs (miRNAs), 21–22-nucleotide secondary siRNAs and 24-nucleotide heterochromatic siRNAs (hetsiRNAs). All small RNAs in plants are modified at their 3′-end by 2′- O -methylation, including miRNAs, which lack this modification in animals. This modification confers stability and protection from degradation. Plant miRNAs are mainly involved in post-transcriptional gene silencing (PTGS) by transcript cleavage or translational repression, and also trigger secondary siRNA production from RNA polymerase II (Pol II) transcripts. Secondary small RNAs of 21 and 22 nucleotides in length are involved in cleavage or translational repression of target transcripts in cis and in trans . They are also able to initiate TGS by establishing DNA methylation at particular loci. The majority of siRNAs in plants are 24-nucleotide hetsiRNAs and are involved in silencing repeats and transposable elements by RNA-directed DNA methylation (RdDM). Small RNAs in plants are involved in reproductive transitions, including meiosis and gametogenesis, and regulate important epigenetic mechanisms such as genomic imprinting and paramutation. Plant genomes encode diverse small RNAs, such as microRNAs, secondary siRNAs, heterochromatic siRNAs and various RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins. Together, these constitute several genetic and epigenetic silencing pathways with diverse cellular and developmental functions, in processes including reproductive transitions, genomic imprinting and paramutation. Plant genomes encode various small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small-RNA classes varies among plant species, suggesting coevolution between environmental adaptations and gene-silencing mechanisms. Biogenesis of small RNAs in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary siRNAs and heterochromatic siRNAs, and their diverse cellular and developmental functions, including in reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small-RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins.

An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

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. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg 2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAG AGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. Cryo-EM reveals the configuration of substrate pre-mRNA within the active spliceosome and suggests how remodelling occurs prior to exon ligation. Structure of the branched splicing complex The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.Barucci et al. show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is mediated by the epigenetic silencing of all of the replicative histone genes.

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein–protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein–protein and RNA–RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA–RNA assembly in cells could lead to disease.

RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing — by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade.The potential of therapeutically targeting RNA structures with small molecules is being increasingly recognized. Here, Disney and colleagues review strategies to identify, validate and optimize small-molecule RNA binders. Examples of existing RNA-targeted small molecules, as well as challenges and future directions in the field, are discussed.

Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.

Transcriptome-wide mapping of N1-methyladenosine (m 1 A) at single-nucleotide resolution reveals m 1 A to be scarce in cytoplasmic mRNA, to inhibit translation, and to be highly dynamic at a single site in a mitochondrial mRNA. The basis of m1A modification N 1 -methyladenosine (m 1 A) modification has been detected on mRNA, but validation of the internal mRNA sites at which it occurs and the functional consequences of it have not been well defined. Schraga Schwartz and colleagues now address these limitations using a method that enables single-nucleotide resolution of such sites in the transcriptome. They show that the level of modification is much lower than reported previously and varies during development and by tissue type. The authors identify a structural motif associated with the modification and define the enzymatic machinery responsible for the methylation. They find that m 1 A modification is associated with translational repression, consistent with its tight regulation. Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N 1 -methyladenosine (m 1 A), which disrupts Watson–Crick base pairing, at internal sites of mRNAs 1 , 2 . These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m 1 A at single-nucleotide resolution. Within the cytosol, m 1 A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m 1 A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m 1 A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m 1 A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m 1 A levels was adopted as a potential means of post-transcriptional regulation.