Exploration of small RNAs

For several decades, only a limited number of noncoding RNAs, such as ribosomal and transfer RNA, have been studied in any depth. In recent years, additional species of noncoding RNAs have increasingly been discovered. Of these, small RNA species attract particular interest because of their essential roles in processes such as RNA silencing and modifications. Detailed analyses revealed several pathways associated with the function of small RNAs. Although these pathways show evolutional conservation, there are substantial differences. Advanced technologies to profile RNAs have accelerated the field further resulting in the discovery of an increasing number of novel species, suggesting that we are only just beginning to appreciate the complexity of small RNAs and their functions. Here, we review recent progress in novel small RNA exploration, including discovered small RNA species, their pathways, and devised technologies.     

The balance of power in RNA trafficking

In the past two years, several different RNA trafficking pathways have been characterized in oligodendrocytes; similar trafficking pathways have been discovered in neuronal and retroviral systems; co-assembly of multiple different RNAs into the same granules has been analyzed as a mechanism for coordinating gene expression; and a new hypothesis for RNA trafficking, based on the balance of power between kinesin and dynein in individual RNA granules, has been proposed.     

RNA under attack: Cellular handling of RNA damage

Damage to RNA from ultraviolet light, oxidation, chlorination, nitration, and akylation can include chemical modifications to nucleobases as well as RNA-RNA and RNA-protein crosslinking. In vitro studies have described a range of possible damage products, some of which are supported as physiologically relevant by in vivo observations in normal growth, stress conditions, or disease states. Damage to both messenger RNA and noncoding RNA may have functional consequences, and work has begun to elucidate the role of RNA turnover pathways and specific damage recognition pathways in clearing cells of these damaged RNAs.     

Viral induction and suppression of RNA silencing in plants

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

RNA interference in fungi: pathways, functions, and applications

Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.     

Small RNA-mediated gene silencing pathways in C. elegans

Small RNA pathways, including the RNA interference (RNAi) pathway and the microRNA (miRNA) pathway, regulate gene expression, defend against transposable elements and viruses, and, in some organisms, guide genome rearrangements. The nematode Caenorhabditis elegans (C. elegans) has been at the forefront of small RNA research; not only were the first miRNAs and their function as regulators of gene expression discovered in C. elegans, but also double-stranded RNA-induced gene silencing by RNAi was discovered in this model organism. Since then, genetic and RNAi-mediated screens, candidate gene approaches, and biochemical studies have uncovered numerous factors in the small RNA pathways and painted a rich palette of interacting pathways. Here we review the different small RNAs that have been discovered in C. elegans and discuss our understanding of their biogenesis pathways and mechanisms of action.     

Effects and side-effects of viral RNA silencing suppressors on short RNAs

In eukaryotes, short RNAs play a crucial regulatory role in many processes including development, maintenance of genome stability and antiviral responses. These different but overlapping RNA-guided pathways are collectively termed 'RNA silencing'. To counteract an antiviral RNA silencing response, plant viruses express silencing suppressor proteins. Recent results have shown that silencing suppressors operate by modifying the accumulation and/or activity of short RNAs involved in the antiviral response. Because RNA silencing pathways intersect, silencing suppressors can also inhibit other short-RNA-regulated pathways. Thus, suppressors contribute to viral symptoms. These findings fuel further research to test whether certain symptoms caused by animal viruses are also manifestations of altered RNA regulatory pathways.     

Small noncoding RNAs in the germline

Small noncoding RNAs have emerged as potent regulators of gene expression, especially in the germline. We review the biogenesis and regulatory function of three major small noncoding RNA pathways in the germline: The small interfering RNA (siRNA) pathway that leads to the degradation of target mRNAs, the microRNA (miRNA) pathway that mostly represses the translation of target mRNAs, and the newly discovered Piwi-interacting RNA (piRNA) pathway that appears to have diverse functions in epigenetic programming, transposon silencing, and the regulation of mRNA translation and stability. The siRNA and miRNA pathways are present in the germline as well as many somatic tissues, whereas the piRNA pathway is predominantly confined to the germline. Investigation of the three small RNA pathways has started to reveal a new dimension of gene regulation with defining roles in germline specification and development.     

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotie RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved time-tional amino acids between archaeal and eukaryotic Piwi domains suggested their functional similarity. Our results provide new clues to the evolution of RNA silencing pathways.     

Biochemical specialization within Arabidopsis RNA silencing pathways

In plants, the RNA silencing machinery responds to numerous inputs, including viral infection, microRNAs, and endogenous siRNAs that may act both in trans and in cis. Additionally, the full spectrum of silencing outcomes has been demonstrated in plants, ranging from mRNA degradation to repression at the level of protein synthesis to chromatin remodeling. Genetic studies in Arabidopsis have indicated that individual response pathways are functionally compartmentalized. However, to date, no biochemical systems have been available to investigate the roles of specific proteins within silencing pathways or the effects of selected mutations on the biochemical activity of those components. Here, we describe the generation of Arabidopsis extracts that reproduce many aspects of RNA silencing reactions in vitro. We find that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNA silencing pathways in Arabidopsis.     

Ancient and Novel Small RNA Pathways Compensate for the Loss of piRNAs in Multiple Independent Nematode Lineages

Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.     

Taking RISCs with Ago hookers

Argonautes are central and common components of crucial effectors of RNA silencing pathways. Although earlier steps in these pathways, such as small RNA biogenesis and their loading into AGO, have been quite well described, our knowledge on regulation of the action of AGO and their partners is still poor. Recent breakthroughs have highlighted the existence in many eukaryotes of an evolutionarily conserved motif, the Ago-hook, in factors implicated in AGO action. Furthermore, it has been shown that certain plant pathogen proteins have co-opted the Ago-hook as a means of evasion of plant defense systems. Here we discuss the roles and properties of Ago-hook proteins in divergent RNAi-related pathways.