Research
The main objective of our lab is to understand epigenetic mechanisms underlying host-parasite interactions. We are particularly interested in neglected tropical parasites.
Our research focuses on understanding how small RNAs regulate the parasite's lifecycle and pathogenesis and influence host-pathogen interaction. Also, our research will focus on understanding the biogenesis of the parasite small RNAs by atypical parasite small RNA machinery and the influence of stress and host factors on small RNA production and their function.
Why Protozoan Parasites?
Even though proteins involved in the RNAi pathway are well conserved, there is a degree of plasticity introduced by the presence of multiple homologs of DICER or AGOs depending on the presence of different types of sRNAs in each species and their functions. However, there are exceptions to this conservation, with unicellular protozoa like Plasmodium falciparum among others lacking the RNAi pathway (Baum et al 2009). Certain parasitic protozoa have been shown to possess either siRNAs or miRNAs (Kolev 2011), with a proposed role of silencing transposons and repeats in Trypanosoma brucei (Kolev 2011) and Toxoplasma gondii (Braun 2010) and silencing of coding regions in Entamoeba (Zhang et al 2008. 2021). A common feature of the RNAi pathway in these organisms is the lack of canonical RNAi factors (Kolev 2011). Both DICER and AGO in most of these protozoa lack various canonical domains including the helicase and dsRNA binding domains in DICER or the conserved PAZ or MID domains involved in sRNA binding in AGOs. Despite their discovery, how sRNAs are produced using these atypical RNAi factors in vivo, and their function in these neglected protozoan parasites remain to be investigated.
Therefore, to understand the plasticity and evolution of the RNAi pathway and its function in protozoan parasites, we will investigate the sRNA biogenesis and epigenetic regulation of pathogenesis by comparative analysis of the pathway in these neglected, yet clinically relevant protozoan parasites. We are also interested in developing diagnostics and intervention studies as an extension of our fundamental interests in host-parasite interaction.
The three main questions include
1. How small RNAs are synthesized using minimal and atypical RNAi machinery?
2. What is the role of the small RNA pathway in the life cycle of parasites?
3. How small RNAs regulate host-pathogen interaction?
We are using state-of-the-art tools which we are constantly evolving based on our questions and requirements. These include:
small RNA and mRNA sequencing (Singh et al 2021a, b)
measurement of Translation by Ribo-seq (Singh et al 2021a)
Global Run on Sequencing ( to measure transcription) (Singh et al 2021a, Barucci et al 2020)
individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation & sequencing (iCLIP-seq)
Proteomics, biochemical and molecular tools to analyze protein-protein and RNA-protein interactions
References
Zhang, H., Ehrenkaufer, G. M., Pompey, J. M., Hackney, J. A. & Singh, U. Plos Pathog 4, e1000219 (2008).
Pappas, G., Roussos, N. & Falagas, M. E. Int J Parasitol 39, 1385–1394 (2009).
Baum, J. et al. Nucleic Acids Res 37, 3788–3798 (2009).
Braun, L. et al. Plos Pathog 6, e1000920 (2010).
Kolev, N. G., Tschudi, C. & Ullu, E. Eukaryot Cell 10, 1156–1163 (2011).
Singh, S., Munawwar, A., Rao, S., Mehta, S. & Hazarika, N. K. Plos Neglect Trop D 8, e2737 (2014).
Singh, M. et al. Nat Commun 12, 3492 (2021a).
Singh, M. et al. Embo Rep 23:e54341 (2021b).
Barucci, G. et al. Nat Cell Biol 22, 1–11 (2020).
Zhang, H., Veira, J., Bauer, S. T., Yip, C. & Singh, U. Mbio 12, e01540-21 (2021).