Helge Großhans

Active turnover modulates mature microRNA activity in Caenorhabditis elegans

MicroRNAs (miRNAs) constitute a large class of regulatory RNAs that repress target messenger RNAs to control various biological processes. Accordingly, miRNA biogenesis is highly regulated, controlled at both transcriptional and post-transcriptional levels, and overexpression and underexpression of miRNAs are linked to various human diseases, particularly cancers. As RNA concentrations are generally a function of biogenesis and turnover, active miRNA degradation might also modulate miRNA accumulation, and the plant 3′→5′ exonuclease SDN1 has been implicated in miRNA turnover. Here we report that degradation of mature miRNAs in the nematode Caenorhabditis elegans, mediated by the 5′→3′ exoribonuclease XRN-2, affects functional miRNA homeostasis in vivo. We recapitulate XRN-2-dependent miRNA turnover in larval lysates, where processing of precursor-miRNA (pre-miRNA) by Dicer, unannealing of the miRNA duplex and loading of the mature miRNA into the Argonaute protein of the miRNA-induced silencing complex (miRISC) are coupled processes that precede degradation of the mature miRNA. Although Argonaute:miRNA complexes are highly resistant to salt, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by the addition of miRNA target RNA. Our results therefore suggest the presence of an additional layer of regulation of animal miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions and for the maintenance of steady-state concentrations of miRNAs. This pathway might represent a potential target for therapeutic intervention on miRNA expression.

Molecular biology: the expanding world of small RNAs.

Molecular cell biology has long been dominated by a protein-centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate gene expression at different levels, and have essential roles in health and disease. Small RNAs, non-coding RNAs consisting of 20 to 30 nucleotides, regulate many biological processes. There may be tens of thousands of such regulatory RNAs in the mammalian genome. What do they all do? This Q&A session gets the general reader up to speed.

MicroRNA turnover: when, how, and why

MicroRNAs (miRNAs) are short (∼22 nucleotide) RNAs that are important for the regulation of numerous biological processes. Accordingly, the expression of miRNAs is itself tightly controlled by mechanisms acting at the level of transcription as well as processing of miRNA precursors. Recently, active degradation of mature miRNAs has been identified as another mechanism that is important for miRNA homeostasis. Here we review the molecular factors and cellular conditions that promote miRNA turnover. We also discuss what is known about the physiological relevance of miRNA decay.

Repression of C. elegans microRNA targets at the initiation level of translation requires GW182 proteins

MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell‐free and cell‐based assays have supported the idea that miRNAs repress their target mRNAs by blocking initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let‐7 miRNA, an important regulator of stem cell fates. We report that let‐7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin‐4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN‐1 and AIN‐2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms.

Regulating the regulators: mechanisms controlling the maturation of microRNAs

MicroRNAs (miRNAs) are small noncoding RNAs that control diverse cellular and developmental events through repression of large sets of target mRNAs. Regulated transcription of the genes encoding miRNAs by RNA polymerase II promotes specific expression patterns of individual miRNAs. However, recent studies have established that substantial regulation of mature miRNA accumulation also occurs after transcription. Here, we review the mechanisms of such post-transcriptional regulation, with a particular focus on examples where molecular mechanisms or physiological principles are beginning to emerge. Elucidating these mechanisms will increase our understanding of gene regulation and provide new insights into causes of miRNA misexpression in diseases such as cancer.

Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans

We have achieved targeted heritable genome modification in Caenorhabditis elegans by injecting mRNA of the nuclease Cas9 and Cas9 guide RNAs. This system rapidly creates precise genomic changes, including knockouts and transgene-instructed gene conversion.

A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p

The yeast ubiquitin‐protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis. Here, we identify Rsp5p in a screen for tRNA export (tex) mutants. The tex23‐1/rsp5‐3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre‐ribosomal subunits. In contrast, nuclear localization sequence (NLS)‐mediated nuclear protein import is unaffected in this mutant. Strikingly, the nuclear RNA export defects seen in the rsp5‐3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast. These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways.

Extensive Oscillatory Gene Expression during C. elegans Larval Development

Oscillations are a key to achieving dynamic behavior and thus occur in biological systems as diverse as the beating heart, defecating worms, and nascent somites. Here we report pervasive, large-amplitude, and phase-locked oscillations of gene expression in developing C. elegans larvae, caused by periodic transcription. Nearly one fifth of detectably expressed transcripts oscillate with an 8 hr period, and hundreds change >10-fold. Oscillations are important for molting but occur in all phases, implying additional functions. Ribosome profiling reveals that periodic mRNA accumulation causes rhythmic translation, potentially facilitating transient protein accumulation as well as coordinated production of stable, complex structures such as the cuticle. Finally, large-amplitude oscillations in RNA sampled from whole worms indicate robust synchronization of gene expression programs across cells and tissues, suggesting that these oscillations will be a powerful new model to study coordinated gene expression in an animal.

The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila

MicroRNA (miRNA) biogenesis proceeds from a primary transcript (pri‐miRNA) through the pre‐miRNA into the mature miRNA. Here, we identify a role of the Caenorhabditis elegans nuclear export receptor XPO‐1 and the cap‐binding proteins CBP‐20/NCBP‐2 and CBP‐80/NCBP‐1 in this process. The RNA‐mediated interference of any of these genes causes retarded heterochronic phenotypes similar to those observed for animals with mutations in the let‐7 miRNA or core miRNA machinery genes. Moreover, pre‐ and mature miRNAs become depleted, whereas primary miRNA transcripts accumulate. An involvement of XPO‐1 in miRNA biogenesis is conserved in Drosophila, in which knockdown of Embargoed/XPO‐1 or its chemical inhibition through leptomycin B causes pri‐miRNA accumulation. Our findings demonstrate that XPO‐1/Emb promotes the pri‐miRNA‐to‐pre‐miRNA processing and we propose that this function involves intranuclear transport and/or nuclear export of primary miRNAs.