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Ever since microRNAs (miRNAs) were first recognized as an extensive gene family >20 years ago, a broad community of researchers was drawn to investigate the universe of small regulatory RNAs. Although core features of miRNA biogenesis and function were revealed early on, recent years continue to uncover fundamental information on the structural and molecular dynamics of core miRNA machinery, how miRNA substrates and targets are selected from the transcriptome, new avenues for multilevel regulation of miRNA biogenesis and mechanisms for miRNA turnover. Many of these latest insights were enabled by recent technological advances, including massively parallel assays, cryogenic electron microscopy, single-molecule imaging and CRISPR–Cas9 screening. Here, we summarize the current understanding of miRNA biogenesis, function and regulation, and outline challenges to address in the future.In this Review, the authors describe how the application of new technologies to the microRNA (miRNA) field has yielded key insights into miRNA biology. The authors summarize our current understanding of miRNA biogenesis, function and processing, and highlight challenges to address in future research.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal germ line cells, piRNAs have essential roles in germ line development. The first function of PIWI–piRNA complexes to be described was the silencing of transposable elements, which is crucial for maintaining the integrity of the germ line genome. Later studies provided new insights into the functions of PIWI–piRNA complexes by demonstrating that they regulate protein-coding genes. Recent studies of piRNA biology, including in new model organisms such as golden hamsters, have deepened our understanding of both piRNA biogenesis and piRNA function. In this Review, we discuss the most recent advances in our understanding of piRNA biogenesis, the molecular mechanisms of piRNA function and the emerging roles of piRNAs in germ line development mainly in flies and mice, and in infertility, cancer and neurological diseases in humans.PIWI-interacting RNAs (piRNAs) are small non-coding RNAs with essential roles in germ line development through silencing of transposable elements and in regulation of protein-coding genes. Recent studies have deepened our understanding of the biogenesis and function of piRNAs and their roles in infertility, cancer and neurological diseases in humans.

In animals, PIWI-interacting RNAs (piRNAs) of 21–35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N -acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today’s RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions. With six approved drugs, siRNA is now an established therapeutic modality poised for expansion.

Protection against microbial infection in eukaryotes is provided by diverse cellular and molecular mechanisms. Here, we present a comparative view of the antiviral activity of virus-derived small interfering RNAs in fungi, plants, invertebrates and mammals, detailing the mechanisms for their production, amplification and activity. We also highlight the recent discovery of viral PIWI-interacting RNAs in animals and a new role for mobile host and pathogen small RNAs in plant defence against eukaryotic pathogens. In turn, viruses that infect plants, insects and mammals, as well as eukaryotic pathogens of plants, have evolved specific virulence proteins that suppress RNA interference (RNAi). Together, these advances suggest that an antimicrobial function of the RNAi pathway is conserved across eukaryotic kingdoms.

Progress in deciphering the genetic architecture of human sensorineural hearing impairment (SNHI) or loss, and multidisciplinary studies of mouse models, have led to the elucidation of the molecular mechanisms underlying auditory system function, primarily in the cochlea, the mammalian hearing organ. These studies have provided unparalleled insights into the pathophysiological processes involved in SNHI, paving the way for the development of inner-ear gene therapy based on gene replacement, gene augmentation or gene editing. The application of these approaches in preclinical studies over the past decade has highlighted key translational opportunities and challenges for achieving effective, safe and sustained inner-ear gene therapy to prevent or cure monogenic forms of SNHI and associated balance disorders.The authors review genetic studies of sensorineural hearing impairment (SNHI) and their resulting insights into the molecular mechanisms underlying auditory system function. They also discuss preclinical studies of inner-ear gene therapy and key translational opportunities and challenges for treating monogenic forms of SNHI and associated balance disorders.

RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

Key Points A-to-I RNA editing is catalysed by adenosine deaminases acting on RNA (ADARs). Three mammalian ADAR genes ( ADAR1 , ADAR2 and ADAR3 ) with common functional domains have been identified. Protein-coding sequences of a limited number of genes, such as glutamate receptor GRIA2 and serotonin receptor HTR2C , are edited, resulting in dramatic alterations of protein functions. Deficiencies in A-to-I RNA editing cause human diseases and pathophysiology. Genome-wide screening has identified numerous A-to-I editing sites in inverted Alu repeats located in non-coding regions of mRNAs. Alu editing in these transcripts is likely to affect many cellular processes. The biogenesis and function of certain miRNAs is regulated by editing of the primary miRNAs (pri-miRNAs). ADAR1 forms a complex with Dicer to promote the efficacy of miRNA processing and RNA interference (RNAi) in developing embryos. ADAR enzymes convert adenosine to inosine (A-to-I editing) at numerous double-stranded Alu repeats in human transcripts, thereby affecting many cellular processes. Primary microRNAs (miRNAs) are also edited, and ADAR1 directly interacts with Dicer, resulting in the modulation of miRNA expression and activity and of downstream gene expression programmes during embryogenesis. Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.

Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized, most notably microRNAs, small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs associate with Argonaute proteins and, through sequence-specific gene regulation, affect almost every major biological process. Intriguing features of small RNAs, such as their mechanisms of amplification, rapid evolution and non-cell-autonomous function, bestow upon them the capacity to function as agents of intercellular communications in development, reproduction and immunity, and even in transgenerational inheritance. Although there are many types of extracellular small RNAs, and despite decades of research, the capacity of these molecules to transmit signals between cells and between organisms is still highly controversial. In this Review, we discuss evidence from different plants and animals that small RNAs can act in a non-cell-autonomous manner and even exchange information between species. We also discuss mechanistic insights into small RNA communications, such as the nature of the mobile agents, small RNA signal amplification during transit, signal perception and small RNA activity at the destination.Small RNAs (microRNAs, siRNAs, piRNAs and others) function as agents of intercellular communication, particularly in development, reproduction, immunity and inheritance. Chen and Rechavi discuss mechanisms and roles of plant and animal small RNAs in the exchange of information between cells, organisms and even species.

Recognition and repression of RNA targets by Argonaute proteins guided by small RNAs is the essence of RNA interference in eukaryotes. Argonaute proteins with diverse structures are also found in many bacterial and archaeal genomes. Recent studies revealed that, similarly to their eukaryotic counterparts, prokaryotic Argonautes (pAgos) may function in cell defense against foreign genetic elements but, in contrast, preferably act on DNA targets. Many crucial details of the pAgo action, and the roles of a plethora of pAgos with non-conventional architecture remain unknown. Here, we review available structural and biochemical data on pAgos and discuss their possible functions in host defense and other genetic processes in prokaryotic cells. In this review, Aravin and colleagues examine bacterial and archaeal Argonaute proteins, discuss their diverse architectures and their possible roles in host defense, proposing additional functions for Argonaute proteins in prokaryotic cells.