Argonaute navigating the balance between protein translation and small RNA synthesis

First author interview: Dr. Meetali Singh is currently working as a Pasteur-Roux-Cantarini Postdoctoral fellow in the Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris with Dr. Germano Cecere. She obtained her B.Sc. honors degree in Biomedical Sciences from Delhi University and then joined the Indian Institute of Science, Bangalore for the Integrated PhD program where she did her PhD in Prof. Utpal Tatu’s lab at the Department of Biochemistry. During her PhD, Dr. Singh studied the role of the chaperone Hsp90 in the life cycle and virulence of protozoan parasites and investigated the evolution of the Hsp90 complex and its function in primitive protozoa, primarily using parasites like Entamoeba, Trichomonas and Theileria as models. For her post-doc research, Meetali transitioned to explore sRNA-mediated epigenetic gene regulation and inheritance in the free-living worm, C. elegans, in the lab of Dr. Germano Cecere at Institut Pasteur, Paris. She is currently investigating the biogenesis and function of endo-siRNAs and their transmission from soma to germline by integrating proteomic, biochemical, and high-throughput genomic approaches. In her free time, she loves to read, travel and very recently picked up abstract acrylic painting as a hobby.

How would you explain your paper’s key results to the non-scientific community?

Cells of the Reproductive system (germline) have a very important function of transmitting genetic information from parents to offspring. However, recent research has clearly established that in addition to DNA, germ cells also transmit other molecules which determine how the genetic information encoded in the DNA is read and how the mRNA and proteins are produced using this information from the DNA. These molecules which regulate the information provided by the genome (DNA) are part of the Epigenome (derived from Greek word Epi which means “above”, hence epigenome). Epigenetic information includes DNA modification, modification of scaffold proteins involved in the DNA packaging and small RNA (sRNA) molecules. In our current study, we focused on sRNAs using the roundworm, Caenorhabditis elegans as a model. Apart from transmitting the life experience from parent to offspring, sRNAs also stop the expression of deleterious genes and provide defence against foreign elements in the germline to maintain genome integrity.

However, in C. elegans germline, thousands of sRNAs are produced against self-germline genes. These sRNAs are loaded by a specialized protein called CSR-1. Mutation of CSR-1 leads to embryonic lethality highlighting its importance, but there has been a lot of confusion about what exactly is the role of the CSR-1-sRNA pathway and how they are produced? In the current study, we address the function of this mysterious sRNA pathway and found it to have dual functions. First, it is involved in regulating the expression of few germline genes by virtue of its RNA-slicing activity and second, it can protect genes involved in oocyte (egg) formation from piRNA mediated silencing. These two functions can co-exist as CSR-1 localizes in the cytoplasm where it can slice the mRNA, and additionally it also localizes in specialized phase-separated liquid droplets (imagine oil droplets in water) called germ granules where it has been proposed to prevent piRNA mediated silencing of germline genes (Figure 1).

Figure 1: The image shows the germline of C. elegans (outlined with dotted line). CSR-1 tagged with mCherry (Red) is present in the cytosol and peri-nuclear structures called germ-granules (marked by arrow). This strain also expresses a germ granule marker tagged with GFP (in green), therefore, when CSR-1 overlaps with germ granule we see a yellow signal.

Interestingly, we found that the major impact of CSR-1 slicing of the germline mRNAs is for the biogenesis of the sRNAs. Slicing of mRNA by CSR-1 leads to the recruitment of the enzyme responsible for producing sRNAs from the sliced mRNAs. The important question here is how does this enzyme know how many sRNAs it needs to make from each mRNA target of CSR-1? What we found is that CSR-1 sRNAs are produced from the mRNAs which are engaged in translation (process of making protein from mRNA). If the mRNA is highly engaged in translation there are fewer sRNAs and if the translation for an mRNA is low, more sRNAs are produced and loaded by CSR-1 (Figure 2). This balance of sRNA levels loaded by CSR-1 is very important as the mRNAs which have very high levels of sRNAs loaded by CSR-1 are the ones degraded by CSR-1.

This work is a result of amazing collaborations with members of the Cecere lab and the Institut Curie mass spectrometry facility.

Figure 2: Model illustrating biogenesis of CSR-1-sRNA in the cytosol on translating mRNA. CSR-1 cleave the mRNA downstream of ribosome to recruit the enzyme which produces sRNA (EGO-1). Ribosome occupancy determines amount of sRNA produced.

In the current study, we focused on understanding the molecular mechanisms involved in the biogenesis of sRNAs.

What are the possible consequences of these findings for your research area?

In the current study, we focused on understanding the molecular mechanisms involved in the biogenesis of sRNAs. These CSR-1 associated sRNAs, once produced in the germline are inherited by the offspring, where they are involved in degrading maternally inherited mRNAs during maternal-to-zygotic transition (a process where maternal mRNAs are degraded, and the offspring starts to transcribe its own mRNAs) (Quarato et al. 2021). These functions of the CSR-1 in germline and embryo makes it an essential factor for fertility and embryonic development, despite this, till now there was no clarity on how CSR-1 sRNAs were produced. We addressed this open question and show that sRNA biogenesis is regulated by the translational status of the target mRNA. This is specifically interesting to further understand the regulation by CSR-1sRNA pathway and more importantly, we propose this mechanism can be conserved across species as there are reports from plant and mouse models which also show an interplay of sRNA biogenesis and translation.

What was the exciting moment (eureka moment) during your research?

There have been many eureka moments throughout the course of this study. But if I must pick one, I will say it was one nice Parisian summer afternoon in 2019 when we were analysing the sequencing data from a Ribo-seq experiment and found the link between sRNA biogenesis and translation. That evening steered us in a totally new direction leading to the publication of this paper.

What do you hope to do next?

Next, I am planning to start my own research group where I would like to focus on the epigenetic mechanisms employed by parasites for their survival in the hostile host environment and regulate their life cycles in the host. These studies can help us understand the mechanism of disease pathogenesis and provide us with potential targets for developing new therapies.

Where do you seek scientific inspiration?

I am a curious person by nature and my family has a big role in instilling this level of curiosity. I seek inspiration from the amazing discussions I have with my peers. I have been lucky to have wonderful teachers during my undergrad days and internships and amazing mentors during my PhD at IISc and now my postdoc at Institut Pasteur. They have always been supportive and encouraging. I also feel inspired when communicating science with people from other backgrounds and young students as they ask very fundamental questions which are often taken for granted by us, people who work in the field, but they make you think deeper.

How do you intend to help Indian science improve?

I intend to start my research lab in India and set up a research program to train a new generation of students to develop scientific temperament. Apart from research-based initiatives, I want to get involved in science communication by collaborating with science illustrators and setting up simplified MOOC courses, seminars for school students and the general public to apprise them of the latest scientific discoveries and research.

Reference

Singh M, Cornes E, Li B, Quarato P, Bourdon L, Dingli F, Loew D, Proccacia S & Cecere G (2021) Translation and codon usage regulate Argonaute slicer activity to trigger sRNAs biogenesis. Nat Commun 12: 3492

Quarato P, Singh M, Cornes E, Li B, Bourdon L, Mueller F, Didier C & Cecere G (2021) Germline inherited sRNAs facilitate the clearance of untranslated maternal mRNAs in C. elegans embryos. Nat Commun 12: 1441

Germano Cecere, PhD: www.cecerelab.com

Edited by: Nikita Nimbark

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