Two microRNAs are sufficient for embryogenesis in C. elegans

 

Background

In 1993, teams led by Gary Ruvkun and Victor Ambros discovered that particular tiny RNAs, which do not encode for proteins, can silence genes with complementary sequences. These tiny RNAs, now known as microRNAs, exist in plants and animals and repress gene expression post-transcriptionally. Biogenesis of metazoan microRNAs is complex: many enzymes and accessory factors are required, in a series of nuclear and cytoplasmic reactions. These reactions turn an RNA Polymerase II transcript (pri-microRNA) into a mature microRNA. Primary microRNA transcripts organize into (at least) one hairpin structure that serves as a substrate for the microprocessor. The microprocessor, composed by the endonuclease Drosha and Pasha/DGCR8, trims the extremities of the primary transcript, leaving only a hairpin (or pre-microRNA). Dicer endonucleases further process the hairpin in the cytoplasm. The mature microRNA guides its Argonaute protein effector partner to partially complementary mRNA targets, ultimately leading to post-transcriptional gene silencing. The depletion of Dicer or Microprocessor in animals leads to highly penetrant embryonic lethality. This implies that at least a subset of microRNAs are essential for development. While a minority of microRNA mutants does show strong developmental defects, a great number of microRNA knock-outs have no discernible phenotype, suggesting more subtle roles in gene regulation. Studies in zebrafish have shown that miR-430 rescues a subset of the developmental defects brought about by Dicer mutation. However, the microRNA or microRNAs required for early embryonic development, and capable of rescuing microprocessor or Dicer defects, have not been identified in other animals. In a recent preprint, Dexheimer and colleagues find that two microRNAs, of the miR-35 and miR-51 families, rescue microprocessor lethality in the nematode Caenorhabditis elegans.

 

Key findings

• The study of the embryonic roles of microprocessor was so far hindered due to the sterility of microprocessor-depleted mothers. To circumvent this, the authors developed a powerful system to conditionally and stringently deplete the microprocessor in embryos, by combining RNAi and auxin-induced degradation of drsh-1 (Drosha) and pash-1 (Pasha/DGCR8) in the maternal germline, a procedure termed RNAiD. No embryos subjected to RNAiD survived beyond morphogenesis. Previous studies have shown that deletion of all the microRNAs of the miR-35 or miR-51 families leads to embryonic arrest. The arrest of RNAiD-treated embryos occurred markedly earlier than in other mutants, including miR-35 or miR-51 whole-family deletions.
• MicroRNAs of the miR-35 and miR-51 families are the most highly expressed during embryogenesis, but in RNAiD-treat embryos only trace amounts of microRNAs are detected.
• Mirtrons are microRNAs originating from introns, whose biogenesis is independent of the microprocessor. The authors constructed an elegant transgenic system with mirtron versions of miR-35 and miR-51 for microprocessor-independent expression. miR-35 and miR-51 mirtrons rescued the embryonic lethality of the miR-35 and miR-51 family deletions, respectively. Importantly, miR-35 and miR-51 mirtrons together could rescue the embryonic lethality of RNAiD and of pash-1 However, these animals did not develop beyond the first larval stage. This suggests that although miR-35 and miR-51 are required for embryogenesis, other microRNAs are required to resume development beyond that point.

 

What I like about this preprint

I like how this paper uses novel, elegant approaches to tackle old problems. MicroRNA researchers know about microprocessor and Dicer embryonic lethality since the early days of microRNAs, but tools to understand these processes in vivo were lacking. Even nowadays, establishing the necessary tools was a challenging and long process, as first author Philipp Dexheimer notes in a Twitter thread describing his work (https://twitter.com/PhilDexheimer/status/1277579653423812610). The mirtron transgenes were a brilliant idea! As miR-35 and miR-51 microRNA families are broadly conserved, this preprint lays the groundwork for future studies addressing the embryonic requirements of homologous microRNAs in other animals.

 

This work is another important reminder that a lot of great biology can still be unraveled using the nematode C. elegans. For small RNAs in particular, the contribution of C. elegans research was tremendous: e.g. it was in C. elegans that microRNAs and RNAi were initially described. It may be tempting to dismiss these little bugs, as invertebrates seem pretty far away from animals more like us, but in doing so, which other broadly relevant insights will we be missing?

 

Open questions

• Are the microRNAs detected in trace amounts in RNAiD embryos mostly mirtrons? Do you think the non-mirtron microRNAs detected in trace amounts may have non-canonical microprocessor-independent biogenesis or do you think they most likely represent contamination/artifact/residual activity of DRSH-1 and PASH-1?
• Have you considered sequencing mRNAs in wild-type, RNAiD, and mirtron-rescued embryos to pinpoint the regulatory dynamics of miR-35 and miR-51 targets? Which genes do you expect to be targeted?
• Do you think such mirtron transgenic approaches can be used to rescue microprocessor mutants in other organisms? Given their conservation, do you expect miR-35 and miR-51 families to be required for embryogenesis in other species?

 

Want to know more?

MicroRNAs: From Mechanism to Organism, Dexheimer & Cochella, 2020

https://www.frontiersin.org/articles/10.3389/fcell.2020.00409/full

 

Metazoan MicroRNAs, Bartel, 2018

https://www.sciencedirect.com/science/article/pii/S0092867418302861

 

A framework for understanding the roles of miRNAs in animal development, Alberti & Cochella, 2017

https://dev.biologists.org/content/144/14/2548.abstract

 

Tags: drosha, embryogenesis, microprocessor, microrna, mir-35, mir-51, mirna, pasha

doi: https://doi.org/10.1242/prelights.23117

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