Work done in the lab of Dr. Purusharth I. Rajyaguru, Associate Professor, at Department of Biochemistry, Indian Institute of Science, Bangalore
About author
Raju Roy completed his Integrated M.Sc in Bioscience and Bioinformatics from the Department of Molecular Biology and Biotechnology, Tezpur University, before joining the Department of Biochemistry, Indian Institute of Science Bangalore as a graduate student in the year 2016 in the laboratory of Dr. Purusharth Rajyaguru. He has also carried out a part of his doctoral thesis research as a visiting Ph.D. student at Institute Curie, Orsay, France, in the laboratory of Dr. Stephan Vagner. His research interest is understanding the dynamics of mRNA-protein complexes in the cell cytoplasm and the regulation of translation in cancer.
Interview
How would you explain your research outcomes to the non-scientific community?
A cell is the smallest structural unit of life and employs various cellular mechanisms to maintain equilibrium. Stress is a common deterrent that affects the normal functioning of the cell. There can be different kinds of stresses that a cell can experience, for example, nutrient deprivation, stress due to DNA damage, exposure to toxic chemicals, etc. Cells deploy various mechanisms and factors to conserve their cellular components and energy during stress. One such mechanism to counter stress is the formation of RNA granules. RNA granules are non-membranous cytoplasmic RNA-protein higher-order complexes (mRNP complexes) form predominantly during any stress conditions. These cytoplasmic complexes disassemble when the cell returns to its normal condition. Two such granules found inside the cell are stress granules (SGs) and processing bodies (P-bodies). Stress granules are thought to be compartments where mRNAs are kept translationally repressed. P-bodies are sites where mRNA present are thought to be awaiting degradation. SGs and PBs are found to be conserved throughout eukaryotes. Previous reports suggested an important role of intrinsically disordered regions (IDRs) during the assembly of RNA granules, which has been extensively studied. IDRs are specific amino acid sequences or repeats in a protein that do not carry a secondary structure. Specifically, the RGG-repeats were found to play an essential role in RNA granule assembly and translation repression. Various mechanisms suggest how these RNA granules form in the cytoplasm; however, the mechanism of the disassembly of RNA granules is still unclear.
Therefore, we were curious to ask how these RNA granules disassemble in the cytoplasm when the stress is removed. We used Saccharomyces cerevisiae as a model system as it is a relatively simple model organism that is easy to work with and can be genetically manipulated in the laboratory. Notably, numerous scientific breakthroughs on several biological processes that were carried out in yeast are found to be true in humans.
We started with a genetic screen where we individually deleted the proteins containing the RGG-repeats in the cell. We noted that the cells with deletion of Sbp1 (Single-strand nucleic acid-binding protein 1) are defective in P-body disassembly. Surprisingly, the RGG-repeats of Sbp1 are found to be essential for the disassembly of P-bodies. Edc3 (Enhancer of decapping protein 3) is a conserved P-body resident protein and has a role in mRNA degradation. We found that Sbp1 can directly bind to Edc3 with the help of its RGG repeats, as the deletion of the IDR abolishes the Edc3-Sbp1 interaction.
Edc3 protein contains a YjeF-N domain known to self-interact (i.e., it can bind to the YjeF-N domain of another Edc3 protein), which is essential for efficient P-body assembly. Our investigation highlighted that the binding of Sbp1 to Edc3 disrupts the Edc3-Edc3 self-interaction, resulting in P-body disassembly.
RNA granules represent multicomponent viscous liquid droplets that form spontaneously by liquid-liquid phase separation (LLPS). Phase-separation can be explained by mixing oil into the water, resulting in the formation of tiny oil droplet-like structures (a process called demixing), which with time fuse to become larger oil droplets in water. Phase-separation of proteins in the cytoplasm follows a similar process of demixing. It has been found that in vitro formation of phase-separated granules can be driven by proteins containing intrinsically disordered regions (IDRs). Therefore, we developed an assay to study the assembly and disassembly of the Edc3 RNA granule in vitro. We found that Edc3 can self-interact and form tiny phase-separated assemblies when added to the phase separation buffer, which grows in size and intensity in the presence of RNA and NADH. When we added Sbp1 in the phase-separated Edc3 assemblies, we found a significant decrease in size and intensity of the assembles, which did not happen when we deleted the IDR of Sbp1, suggesting a direct link between Sbp1 mediated disassembly of Edc3 RNA granule.
It has been implicated that misregulation in the cytoplasmic granule components leads to aggregates. Aberrant granule formation is implicated in various neurodegeneration, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), etc. From our research, we found that low complexity sequences have the potential to disassemble cytoplasmic aggregates of EWSR1, one of the proteins responsible for the progression of neurodegeneration, and can reduce toxicity which is imparted by the persistent aberrant granule in the cell cytoplasm. Our research paved the way for a new focus on specific amino acid sequences, which might be used as a therapeutic intervention against neurodegeneration.
How do these findings contribute to your research area?
Our findings provide three crucial conclusions related to RNA granule biology research. First, we found a disassembly factor that regulates the disassembly of P-bodies, which was previously unknown. Second, the findings assigned a vital role to intrinsically disordered sequences (RGG repeats) in disassembling a specific type of RNA granule. Third, our research suggests a probable mechanism of IDRs and IDR-containing proteins in the dissolution of aberrant granules implicated in the progression of neurodegenerative diseases.
Our research suggests a probable mechanism of IDRs and IDR-containing proteins in the dissolution of aberrant granules implicated in the progression of neurodegenerative diseases.
What was the exciting moment during your research?
As I mentioned earlier, we started our experiment with a screen, and genetic screening can sometimes be tedious. Therefore, there are two exciting moments that I can recall during our research. First, when we found out that Sbp1 might play a role in the disassembly of P-bodies during the screen. Second, we found out that in the absence of Sbp1, the aberrant granules of EWSR1 remain persistent in the cell.
What do you hope to do next?
I am very passionate about asking questions, very unusual questions. Sometimes it leads to a positive result, but many times it backfires. And I think this is what research is all about. I want to explore research in another exciting field of biology, which is the post-transcriptional regulation of mRNA and protein in neurodegeneration and its therapeutic potential. Neurodegeneration is becoming more widespread due to various changing lifestyles and habits. It becomes crucial to study and ask essential questions about the mechanism of changing cellular conditions and what fundamentally leads to the disorder. I hope to continue my research as a post-doctoral fellow in this exciting field of neurobiology.
Where do you seek scientific inspiration from?
I don’t have a particular answer which inspired me to pursue research. First, asking exciting questions after every result, formulating a hypothesis of probable mechanisms associated with the results. Secondly, many people have inspired me since I started my Ph.D. My research advisor, Dr. Purusharth Rajyaguru, always motivated me in every step throughout the research. I am fortunate to get motivated and cheerful lab mates and a friend who never gets tired of scientific and fruitful discussions. Wonderful parents always encourage my research. And most importantly, negative results often led me to endless brainstorming sessions and to be creative to troubleshoot and standardize protocols for any experiment.
How do you intend to help Indian science improve?
Indian science has improved immensely in recent years, though there is always a scarcity of resources. I believe science can only be improved if there is a give and take of knowledge and sharing of expertise, which is still missing. Further, addressing important questions in basic research is still in a latent state. I want to change this culture. I would also try to improve the research environment by conducting more collaborative research, establishing communications within our field of interest, and encouraging researchers to ask more critical and fundamental questions in life sciences.
Reference
Roy, R., Das, G., Kuttanda, I.A. et al. Low complexity RGG-motif sequence is required for Processing body (P-body) disassembly. Nat Commun 13, 2077 (2022). https://doi.org/10.1038/s41467-022-29715-5
Edited by: Vikramsingh Gujar