Linking miRNA export to Extracellular Vesicles – A paradigm change in the understanding of cancer epigenetics

Souvik Ghosh, Kamalika Mukherjee and Yogaditya Chakrabarty‘s joint first author interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Here, authors talk about their work titled “GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of miRNAs In Mammalian Cancer Cells” published in Molecular and Cellular Biology (2021).

Dr. Souvik Ghosh is a Postdoctoral fellow at Biozentrum of the University of Basel, Switzerland. He is exploring diverse RNA mechanisms using advanced Next-Generation Sequencing and Microscopic tools. A passionate nationalist, he is pursuing the journey of taking India to pioneering heights in scientific greatness. As an alumnus of St. Xavier’s Collegiate School, he had the opportunity to walk the same path treaded by the legendary biologist, physicist, botanist, and early writer of science fiction in India, Prof. Jagadish Chandra Bose. His stories, which are a motivation to many, inspired him and gave him the confidence to pursue the journey of science. He went on to study Microbiology in his undergrads and a postgraduation in Biotechnology from the University of Calcutta. Further curiosity in science led him to pursue Ph.D. under the supervision of Dr. S.N. Bhattacharyya at CSIR-IICB Kolkata. With his guidance and interaction, he began the fascinating journey into the world of small RNAs.

A prodigy of Dr. Bhattacharya’s lab, Dr. Kamalika Mukherjee has many accomplishments at such a young age. Awarded with the prestigious INSA Young Scientist Award 2020, she is among the very few recognized and appreciated by Indian National Science Academy. Her initial graduation in chemistry and specialization in organic chemistry during post-graduation from the University of Calcutta has given her the necessary knowledge base to carry out her research work in biology. She pursued her Ph.D. under the supervision of Dr. Suvendra Bhattachryya in his RNA Biology Research Laboratory (RBRL) at CSIR- Indian Institute of Chemical Biology, Kolkata. She has published extensive work on RNA biology, focusing on microRNA-mediated gene regulation especially working on an essential RNA binding protein called HuR. She has explored the role of extracellular vesicles in miRNA export. To her accolades, she has publications in EMBO reports, EMBO molecular medicine, and most recently MCB in 2021 in her first authorship. She has also written a review article in the journal Cytokine. As a postdoctoral fellow, she had training at Institut de Genetique Moleculaire de Montpellier, a CNRS institute in France, where she learned to work on advanced microscopic techniques as well as chromatin immunoprecipitation techniques from Prof. Edouard Bertrand. Presently, she is a research associate at CSIR- Indian Institute of Chemical Biology, Kolkata. She is working on the role of HuR and extracellular vesicles-associated miRNAs in the context of immune response in macrophage cells and the onset of pathogen-induced diseases.

Dr.Yogaditya Chakrabarty is one of the young biotechnologists from India pursuing his dreams at the California Institute of Technology (Caltech), United States. He began his journey as a post-graduate student of Biotechnology at the Institute of Bioinformatics and Biotechnology, Pune University. There he came across the fascinating upcoming field of small RNAs. Under the guidance of Dr. Bhattacharya, interest was invoked in him towards the role of different organellar domains in micro-RNA biogenesis. He decided to pursue his doctoral degree under his guidance. Surroundings define one’s personality, and it’s so true with our young researcher Yogaditya, his personality was groomed, and his vision of science became clearer during his Ph.D. tenure.

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

As per the central dogma, DNA encodes mRNA, which eventually encodes protein via translation. But there are certain RNAs that are non-coding in nature. One such batch of small yet very mighty RNAs, which are barely 20-22nt in size, are the micro RNAs or miRNAs. They bind with the target mRNAs with imperfect complementarity and cause translational repression. These repressed mRNAs are then stored in a translationally dormant state in RNA granules in the cytosol called P-bodies. miRNAs are therefore essential gene-regulating tools of the cells and are present in all cell types. Interestingly, miRNAs level and function get jeopardized in various human diseases, including cancer. The half-life of miRNAs is low in cancer cells, and to find out a plausible reason for this lowering was our initial motto. In this regard, we have found an exciting observation – the target mRNAs are responsible for the miRNA turnover, and the miRNAs, after being engaged in a cycle of translational repression, get exported out of the cells via the extracellular vesicles (EVs) to balance cellular levels of these repressor molecules.

Figure 1. Graphical representation.

EVs are small 100-200nm-sized extracellular vesicles known to carry proteins, RNAs, and even miRNAs and help in cell-cell communication. This substrate mediated miRNA export is specific for a particular miRNA-target mRNA duet, i.e., let-7a is exported via EVs by its target mRNA only. Next, we have identified two candidate molecules or proteins regulating this miRNA export process via EVs. One of them is the TNRC6B/GW182 proteins which restrict this export process by retaining the miRNA-bound RNA in the P-bodies, thereby keeping the cellular miRNA level high. Another important player is the RNA-binding protein HuR which is the miRNA antagonist in function, i.e., it relieves the miRNA-repressed mRNAs and facilitates their translation (Cell, Bhattacharyya et al.,2006). In a previous study (Mukherjee et al., EMBO reports, 2016), we have already reported, in stressed hepatic cells, how HuR binds with the replaced miRNAs reversibly and eventually gets ubiquitinated and decouples from miRNA and facilitates the miRNA export process.

Figure 2. A human cancer cell with GW182 Bodies (purple) and HuR protein (red). The cell actin cytoskeleton is stained with FITC-Phalloidin.

Here, in this study, we found a fascinating observation – HuR facilitates the miRNA export process in an auxiliary pathway of miRNA binding and unbinding. It is independent of the target-driven miRNA export process; unlike GW182 protein, it does not involve P-bodies. Therefore, to cut a long story short, target mRNAs facilitate the export of the previously engaged miRNAs via the EVs. The protein GW182 prevents this EV-mediated export of miRNAs out of the cell whereas another protein HuR promotes this miRNA export via EVs.

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

miRNA export from cells into extravesicular space (or Extracellular vesicles / EV) opens a new paradigm for biomarker discovery. It is attractive for the identification of new targets for liquid biopsies. However, it also points to the importance of defining new standards for novel cell-specific RNA signatures. This research work proposes a feed-forward loop where the level of a particular miRNA within a cancer cell is regulated by its target mRNA. Let-7a is an anti-proliferative miRNA that induces cell senescence. Therefore, if the EV-mediated export of let-7a miRNAs can be blocked, then the accumulation of let-7a within cells may lead to an increase in the percentage of senescent cells. EV-mediated export can be blocked either by preventing the EV formation, i.e., by using the drug GW4869 or by ectopically expressing GW182 protein or by depleting or inhibiting the protein HuR. If one or more of these can be applied along with the anti-cancer drugs, it may open new and more effective avenues for the treatment of cancer by drugs like geldanamycin.

Moreover, as mentioned above, extracellular vesicles or EVs are already considered important for non-invasive detection or diagnosis of diseases like cancer as EVs can be isolated from blood serum, plasma, urine, milk, or even saliva by standardized methods. Their protein, RNA, and even miRNA levels can be measured reliably and compared with known disease markers to get an idea of disease progression or onset.

After being exported out of a cell, miRNAs can also be transferred to other cells, thereby rescuing the miRNA level in the recipient cell and maintaining proper miRNA homeostasis in donor cells. EV-associated miRNAs may function like hormones carrying important gene-regulatory messages and signals to even other cell types near or far, thereby playing a pivotal role in cell-cell communication.

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

Souvik– The observation that mRNAs can also downregulate their regulators, i.e., the miRNAs, was surprising. However, it was also exciting since it challenged the overwhelming notion that the only role of miRNAs was to downregulate their target mRNAs. We observed a reciprocal regulation, not totally unheard of in science but definitely intriguing to the small RNA scientific community. To note that miRNA interaction partners like GW182 or HuR also play a role in this downregulation was mystifying, but it also spurred us on to new avenues of investigation.

Kamalika-The observation that cellular miRNA level of pre-engaged miRNAs are getting lowered as they are packaged explicitly into extracellular vesicles and eventually exported out as parcel or cargo, and this phenomenon is specifically facilitated/ mediated by their own target mRNA – this was exciting!!

Also, to identify the candidate molecule (GW182 protein), which prevents this target-driven miRNA export via EVs and delineate its mechanism by scoring the vital role of P-bodies in this complex mechanism – this was very interesting!

Lastly, to find out the role of HuR in facilitating the EV-mediated miRNA export and its crosstalk with GW182. We had already reported the role of the HuR protein and delineated the mechanism in detail earlier, in two different contexts, in our previous publications in EMBO reports and EMBO Mol Med, respectively (both are highly cited and praised by other researchers already). Here we found interesting observation that completed the picture and solved the mechanistic jigsaw puzzle. We found HuR facilitates the EV-mediated miRNA export from cancer cells, and this phenomenon is independent of GW182 protein level, P-bodies, and substrate mRNA – therefore, it’s an auxiliary pathway!! (This gave us goosebumps!!)

Yogaditya– The fact that micro-RNAs are specifically packaged and exported outside the cells amidst such rigorous modulation by protein factors such as GW182 and HuR is very thought-provoking. On top of that, there are additional regulation levels in the form of different spatial compartments and intracellular dynamics. Overall, it presents an intriguing scenario worth further dissection.

What do you hope to do next?

Souvik– I am motivated to further explore the concepts of miRNAs that I have acquired while working in the lab of Dr. S.N Bhattacharyya (as a Doctoral candidate), and also subsequently during my Post-Doc tenure at the University of Basel (Switzerland). I hope to bring the whole concept to the bedside as a therapeutic tool (i.e., to the patients). I also wish to mobilize the use of miRNAs as diagnostic tools to detect and monitor diseases, including cancer. Detection of the extracellular RNA through rapid and non-invasive methods has immense potential yet to be tapped.

Kamalika– After working extensively on miRNA mediated gene regulation and RNA binding proteins along with extracellular vesicles. I now want to shift gears a bit and use my knowledge of chemistry (as I have done my masters in organic chemistry) along with my expertise in molecular biology to venture into the world of chemical biology. I want to work on designing and functioning of useful RNA inhibitors in the context of EVs. I want to focus on basic science research as it’s my passion. On the other hand, being the humble winner of the prestigious INSA Young Scientist award presented by the Indian National Science Academy, New Delhi, I feel it’s also my responsibility to contribute something effective in translational research. I had also worked as a short postdoctoral fellow at Institut de Genetique Moleculaire de Montpellier, a CNRS institute in France where I had the opportunity to learn advanced microscopic techniques from Prof. Edouard Bertrand, a pioneer in single molecular RNA imaging. I want to use that training and my biochemistry research experience to work on various fascinating research questions.

Yogaditya– With my current research focusing on mitochondrial dynamics, I hope to define factors responsible for the spatial isolation of mitochondrial metabolic defects and their role in many genetic disorders. I want to carry these thoughts further to an independent academic position.

Where do you seek scientific inspiration?

Souvik– I seek inspiration from the sayings of the great sage of India, Swami Vivekananda who says, “Arise, awake, and stop not till the goal is reached.” By being honest and sincere in the work I do, I would believe that I have served the Almighty.

Kamalika– I have always been fascinated by science. Research is my passion, and a new experiment or any interesting data gives me goosebumps. I am fortunate to be married to my Ph.D. supervisor Prof. Suvendra Bhattacharyya, a senior scientist and a Shanti Swarup Bhatnagar Awardee. We keep discussing science and research 24×7, and this keeps both of us motivated. My parents are my constant source of inspiration. Suvendra inspires me, not only with his achievements, awards and publications but also with the amount of hard work, patience, perseverance, and tremendous effort he puts in achieving them.

Yogaditya– My inspiration comes from my imagination, and my vision is fueled by the world around me.

How do you intend to help Indian science improve?

Souvik– Indian science is at the threshold of greatness. To reach the pinnacle, I would like to contribute to science administration if given the opportunity. With rigorous training in entrepreneurial and project management skills, I would like to see the implementation of a robust scientific temperament, structured on the pillars of professional ethics, free from malignancies of misused power, ego and finances that have long plagued the development of India as a scientific powerhouse.

Kamalika– Indian science is a gold-mine with so many masterminds and enthusiastic young researchers all around. We need to make our efforts a little more organized and maintain a perfect balance between basic research and translational research. Also, we have to take the initiative and come up with new challenging, and interesting research ideas and open new avenues for collaborations and network projects using our human resources and expertise to its fullest extent.

Yogaditya– Indian science has enormous potential, but it is still mired in egocentric hierarchical systems. To reach our desired heights, we need to dissolve the hierarchy to a great extent and sate our egos through discussion. There are few centers following these ideologies, but these are few and far between. Given the opportunity and my limited prowess in science administration, I would like to contribute to garnering more of such centers in whatever capacity possible.

Reference

Souvik Ghosh, Kamalika Mukherjee, Yogaditya Chakrabarty, Susanta Chatterjee, Bartika Ghoshal, Suvendra N. Bhattacharyya. GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of miRNAs In Mammalian Cancer Cells. Molecular and Cellular Biology Mar 2021, MCB.00483-20; DOI: 10.1128/MCB.00483-20

Email: souvik1983@gmail.com; mb.kamalika@gmail.com; yogaditya.c@gmail.com

Prof. Suvendra Bhattacharyya lab: https://rnabiolab.wixsite.com/suvendralab

Edited by: Dolly Singh and Manveen K. Sethi (Copy Editor, Volunteer, Biopatrika)

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