Expression, not sequence, distinguishes miR-238 from its miR-239ab sister miRNAs in promoting longevity in Caenorhabditis elegans.

MicroRNAs (miRNAs) regulate gene expression by base-pairing to target sequences in messenger RNAs (mRNAs) and recruiting factors that induce translational repression and mRNA decay. In animals, nucleotides 2-8 at the 5' end of the miRNA, called the seed region, are often necessary and sometimes sufficient for functional target interactions. MiRNAs that contain identical seed sequences are grouped into families where individual members have the potential to share targets and act redundantly. A rare exception seemed to be the miR-238/239ab family in Caenorhabditis elegans, as previous work indicated that loss of miR-238 reduced lifespan while deletion of the miR-239ab locus resulted in enhanced longevity and thermal stress resistance. Here, we re-examined these potentially opposing roles using new strains that individually disrupt each miRNA sister. We confirmed that loss of miR-238 is associated with a shortened lifespan but could detect no longevity or stress phenotypes in animals lacking miR-239a or miR-239b, individually or in combination. Additionally, dozens of genes were mis-regulated in miR-238 mutants but almost no gene expression changes were detected in either miR-239a or miR-239b mutants compared to wild type animals. We present evidence that the lack of redundancy between miR-238 and miR-239ab is independent of their sequence differences; miR-239a or miR-239b could substitute for the longevity role of miR-238 when expressed from the miR-238 locus. Altogether, these studies disqualify miR-239ab as negative regulators of aging and demonstrate that expression, not sequence, dictates the specific role of miR-238 in promoting longevity.

A rADAR defense against RNAi.

Adenosine deaminases that act on RNA (ADARs) convert adenosines (A) to inosines (I) in stretches of dsRNA. The biological purpose of these editing events for the vast majority of ADAR substrates is largely unknown. In this issue of Genes & Development, Reich and colleagues (pp. 271-282) demonstrate that in Caenorhabditis elegans, A-to-I editing in double-stranded regions of protein-coding transcripts protects these RNAs from targeting by the RNAi pathway. Disruption of this safeguard through loss of ADAR activity coupled with enhanced RNAi results in developmental abnormalities and profound changes in gene expression that suggest aberrant induction of an antiviral response. Thus, editing of cellular dsRNA by ADAR helps prevent host RNA silencing and inadvertent antiviral activity.

Pairing beyond the Seed Supports MicroRNA Targeting Specificity.

To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.

Nuclear and cytoplasmic poly(A) binding proteins (PABPs) favor distinct transcripts and isoforms.

The poly(A)-tail appended to the 3'-end of most eukaryotic transcripts plays a key role in their stability, nuclear transport, and translation. These roles are largely mediated by Poly(A) Binding Proteins (PABPs) that coat poly(A)-tails and interact with various proteins involved in the biogenesis and function of RNA. While it is well-established that the nuclear PABP (PABPN) binds newly synthesized poly(A)-tails and is replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A)-tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein-coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A)-tails and PABPC-enriched RNAs had longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A)-tail synthesis, maintenance, and function.

Recovery from heat shock requires the microRNA pathway in Caenorhabditis elegans.

The heat shock response (HSR) is a highly conserved cellular process that promotes survival during stress. A hallmark of the HSR is the rapid induction of heat shock proteins (HSPs), such as HSP-70, by transcriptional activation. Once the stress is alleviated, HSPs return to near basal levels through incompletely understood mechanisms. Here, we show that the microRNA pathway acts during heat shock recovery in Caenorhabditis elegans. Depletion of the miRNA Argonaute, Argonaute Like Gene 1 (ALG-1), after an episode of heat shock resulted in decreased survival and perdurance of high hsp-70 levels. We present evidence that regulation of hsp-70 is dependent on miR-85 and sequences in the hsp-70 3'UTR that contain target sites for this miRNA. Regulation of hsp-70 by the miRNA pathway was found to be particularly important during recovery from HS, as animals that lacked miR-85 or its target sites in the hsp-70 3'UTR overexpressed HSP-70 and exhibited reduced viability. In summary, our findings show that down-regulation of hsp-70 by miR-85 after HS promotes survival, highlighting a previously unappreciated role for the miRNA pathway during recovery from stress.

A sense-able microRNA.

In this issue of Genes & Development, Drexel and colleagues (pp. 2042-2047) present a beautiful example of how microRNAs (miRNAs) can regulate tissue-specific gene expression in a biologically relevant setting. They found that miR-791 is expressed in only three types of carbon dioxide (CO2)-sensing neurons in Caenorhabditis elegans, and its primary function there seems to be repression of two target genes that interfere with the behavioral response to CO2 Interestingly, these two targets are broadly expressed across other tissues. Thus, restricted miRNA expression can lead to target repression in select tissues to promote distinct cellular physiologies.

miRNA Targeting: Growing beyond the Seed.

miRNAs are small RNAs that guide Argonaute proteins to specific target mRNAs to repress their translation and stability. Canonically, miRNA targeting is reliant on base pairing of the seed region, nucleotides 2-7, of the miRNA to sites in mRNA 3' untranslated regions. Recently, the 3' half of the miRNA has gained attention for newly appreciated roles in regulating target specificity and regulation. In addition, the extent of pairing to the miRNA 3' end can influence the stability of the miRNA itself. These findings highlight the importance of sequences beyond the seed in controlling the function and existence of miRNAs.

Remodeling of the Caenorhabditis elegans non-coding RNA transcriptome by heat shock.

Elevated temperatures activate a heat shock response (HSR) to protect cells from the pathological effects of protein mis-folding, cellular mis-organization, organelle dysfunction and altered membrane fluidity. This response includes activation of the conserved transcription factor heat shock factor 1 (HSF-1), which binds heat shock elements (HSEs) in the promoters of genes induced by heat shock (HS). The upregulation of protein-coding genes (PCGs), such as heat shock proteins and cytoskeletal regulators, is critical for cellular survival during elevated temperatures. While the transcriptional response of PCGs to HS has been comprehensively analyzed in a variety of organisms, the effect of this stress on the expression of non-coding RNAs (ncRNAs) has not been systematically examined. Here we show that in Caenorhabditis elegans HS induces up- and downregulation of specific ncRNAs from multiple classes, including miRNA, piRNA, lincRNA, pseudogene and repeat elements. Moreover, some ncRNA genes appear to be direct targets of the HSR, as they contain HSF-1 bound HSEs in their promoters and their expression is regulated by this factor during HS. These results demonstrate that multiple ncRNA genes respond to HS, some as direct HSF-1 targets, providing new candidates that may contribute to organismal survival during this stress.

RNA interference may result in unexpected phenotypes in Caenorhabditis elegans.

RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unexpected phenotypes uncovered by the administration of unspecific exogenous dsRNA. An example involves dsRNA produced by the multiple cloning site (MCS) of L4440, which shares complementary sequences with some widely used reporter vectors and induces partial transgene silencing via the canonical and antiviral RNAi pathway. Going beyond transgene silencing, we found that the reduced embryonic viability of mir-35-41(gk262) mutants is partially reversed by exogenous dsRNA via a mechanism that involves canonical RNAi. These results indicate cross-regulation between different small RNA pathways in C. elegans to regulate embryonic viability. Recognition of the possible unspecific effects elicited by RNAi vectors is important for rigorous interpretation of results from RNAi-based experiments.

Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging.

Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.

Todos Santos small RNA symposium.

Worm biologists from the United States, Canada, and the United Kingdom gathered at the Colorado State University Todos Santos Center in Baja California Sur, Mexico, April 3-5, 2019 for the Todos Santos Small RNA Symposium. Meeting participants, many of whom were still recovering from the bomb cyclone that struck a large swath of North America just days earlier, were greeted by the warmth and sunshine that is nearly ubiquitous in the sleepy seaside town of Todos Santos. With only 24 speakers, the meeting had the sort of laid-back vibe you might expect amongst the palm trees and ocean breeze of the Pacific coast of Mexico. The meeting started with tracing the laboratory lineages of participants. Not surprisingly, the most common parental lineages represented at the meeting were Dr. Craig Mello, Dr. Gary Ruvkun, and Dr. Victor Ambros, whom, together with Dr. Andy Fire and Dr. David Baulcombe, pioneered the small RNA field. In sad irony, on the closing day of the meeting, participants were met with the news of Dr. Sydney Brenner's passing. By establishing the worm, Caenorhabditis elegans, as a model system Dr. Brenner paved the way for much of the research discussed here.

MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship.

MicroRNAs (miRNAs) have emerged as key gene regulators in diverse biological pathways. These small non-coding RNAs bind to target sequences in mRNAs, typically resulting in repressed gene expression. Several methods are now available for identifying miRNA target sites, but the mere presence of an miRNA-binding site is insufficient for predicting target regulation. Regulation of targets by miRNAs is subject to various levels of control, and recent developments have presented a new twist; targets can reciprocally control the level and function of miRNAs. This mutual regulation of miRNAs and target genes is challenging our understanding of the gene-regulatory role of miRNAs in vivo and has important implications for the use of these RNAs in therapeutic settings.

Autoregulation of microRNA biogenesis by let-7 and Argonaute.

MicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65-nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22-nucleotide forms. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in messenger RNA transcripts, leading to translational repression and destabilization of the target messenger RNAs. Here we show that the miRNA complex also targets and regulates non-coding RNAs that serve as substrates for the miRNA-processing pathway. We found that the Argonaute protein in Caenorhabditis elegans, ALG-1, binds to a specific site at the 3' end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive-feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding messenger RNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of autoregulation of let-7 biogenesis establishes a new mechanism for controlling miRNA expression.

Splicing remodels the let-7 primary microRNA to facilitate Drosha processing in Caenorhabditis elegans.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that use partial base-pairing to recognize and regulate the expression of messenger RNAs (mRNAs). Mature miRNAs arise from longer primary transcripts (pri-miRNAs) that are processed to a shorter hairpin precursor miRNA (pre-miRNA) by the Microprocessor complex. In Caenorhabditis elegans the primary let-7 (pri-let-7) transcript undergoes trans-splicing, where pri-let-7 is cleaved at a 3' splice site and the splice-leader-1 (SL1) sequence is appended at the 5' end. Here we investigate the role of this splicing event in the biogenesis of let-7 miRNA. We hypothesized that splicing changes the secondary structure of the pri-let-7 transcript, creating a more favorable substrate for recognition by the Microprocessor. Supporting this idea, we detected conspicuous structural differences between unspliced and SL1-spliced pri-let-7 transcripts using in vitro ribonuclease (RNase) assays. Through the generation of transgenic worm strains, we found that the RNA secondary structure produced by splicing, as opposed to the act of splicing itself, optimizes processing of pri-let-7 by the Microprocessor in vivo. We also observed that the endogenous spliced, but not the unspliced, pri-let-7 transcripts bind to the Microprocessor and accumulate upon its depletion. We conclude that splicing is a key step in generating pri-let-7 transcripts with a structure that enables downstream processing events to produce appropriate levels of mature let-7.

A tale of two sequences: microRNA-target chimeric reads.

In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.

MicroRNA biogenesis: regulating the regulators.

MicroRNAs (miRNAs) function as 21-24 nucleotide guide RNAs that use partial base-pairing to recognize target messenger RNAs and repress their expression. As a large fraction of protein-coding genes are under miRNA control, production of the appropriate level of specific miRNAs at the right time and in the right place is integral to most gene regulatory pathways. MiRNA biogenesis initiates with transcription, followed by multiple processing steps to produce the mature miRNA. Every step of miRNA production is subject to regulation and disruption of these control mechanisms has been linked to numerous human diseases, where the balance between the expression of miRNAs and their targets becomes distorted. Here we review the basic steps of miRNA biogenesis and describe the various factors that control miRNA transcription, processing, and stability in animal cells. The tremendous effort put into producing the appropriate type and level of specific miRNAs underscores the critical role of these small RNAs in gene regulation.

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans.

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we identify the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42(n1089) worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, we propose that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.

The miR-35-41 family of microRNAs regulates RNAi sensitivity in Caenorhabditis elegans.

RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo-RNAi) or natural endogenous (endo-RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo-RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo-siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.

Multiple cis-elements and trans-acting factors regulate dynamic spatio-temporal transcription of let-7 in Caenorhabditis elegans.

The let-7 microRNA (miRNA) is highly conserved across animal phyla and generally regulates cellular differentiation and developmental timing pathways. In Caenorhabditis elegans, the mature let-7 miRNA starts to accumulate in the last stages of larval development where it directs cellular differentiation programs required for adult fates. Here, we show that expression of the let-7 gene in C. elegans is under complex transcriptional control. The onset of let-7 transcription begins as early as the first larval stage in some tissues, and as late as the third larval stage in others, and is abrogated at the gravid adult stage. Transcription from two different start sites in the let-7 promoter oscillates during each larval stage. We show that transcription is regulated by two distinct cis-elements in the promoter of let-7, the previously described temporal regulatory element (TRE), and a novel element downstream of the TRE that we have named the let-7 transcription element (LTE). These elements play distinct and redundant roles in regulating let-7 expression in specific tissues. In the absence of the TRE and LTE, transcription of let-7 is undetectable and worms exhibit the lethal phenotype characteristic of let-7 null mutants. We also identify several genes that affect the transcription of let-7 generally and tissue-specifically. Overall, spatio-temporal regulation of let-7 transcription is orchestrated by multiple cis- and trans-acting factors to ensure appropriate expression of this essential miRNA during worm development.