RNA mediated regulation of reprogramming
Cellular reprogramming is a process opposite of cellular differentiation, wherein a specialized cell is converted into a pluripotent cell that can give rise to different types of cells. Understanding the roadmaps to pluripotency has immense clinical importance. During animal development, a complete reprogramming into pluripotency is rare, being restricted to the process of oocyte to embryo transition (OET). During OET, a fertilized oocyte develops into a pluripotent embryo, which has the potential to give rise to an entire new individual. Chromatin modifiers and transcription factors play essential roles in regulating embryonic genome acitivation (EGA) which is critical for OET. However, more recently several RNA binding proteins have been indentified as cytoplasmic raodblocks to pluripotency by regulating post-transcriptional gene expression. RNAs are regulated extensively after transcription and provide means to fine-tune gene expression both temporally and spatially. RNA binding proteins, by regulating all aspects of RNA metabolism including splicing, polyadenylation, localization, translation and degradation, are central to gene expression. We use C. elegans, a nematode, as a model system to study RNA mediated regulation of pluripotency.
LIN-41, a TRIM-NHL family protein, regulates onset of pluripotency in the C. elegans germline (Tocchini et al., 2014). In additon, LIN-41 also regulates develomental transitions in the soma. Human LIN41 (TRIM71) has been implicated in cellular plasticity, facilitating the reprogramming of differentiated cells into pluripotent cells. LIN-41 functions as an RNA binding protein and induces mRNA decay when bound to the 3’UTR but translational repression when bound to the 5’UTR of somatic mRNAs (Aeschimann et al., 2017). In both cases, the RNA binding is mediated by the NHL domain that, in LIN-41 recognizes a stem-loop RNA element, whose shape determines the RNA-binding specificity
The masters project will be a part of the ongoing project where we aim to identify direct mRNA targets of LIN-41 in the C.elegans germline and uncover the mechanisms underlying LIN-41 mediated RNA regulation. We will study how genes which are post-transcriptionally regulated by LIN-41 control embryonic genome acitvation. As LIN-41 medaited RNA regualtion is conserved in higher oraganisms, we will also use mammalian cell culture system to dissect the molecular circuitary of LIN-41 medaited RNA regulation by transiently expressing the different components in HEK-293 cells.
The student will learn C. elegans genetics, RNA interference, immunoprecipitation, western blotting, microscopy, mammlian cell culture maintenance and transfection. We will also perfrom RNA extraction, qRT-PCR and polyadenylation assays. Most experiments can be inished in a time frame of 1 year.
Selected Pulications –
Aeschimann, F., Kumari, P., Bartake, H., Gaidatzis, D., Xu, L., Ciosk, R., and Grosshans, H. (2017). LIN41 Post-transcriptionally Silences mRNAs by Two Distinct and Position-Dependent Mechanisms. Molecular cell 65, 476-489 e474.
Kumari, P., Aeschimann, F., Gaidatzis, D., Keusch, J.J., Ghosh, P., Neagu, A., Pachulska-Wieczorek, K., Bujnicki, J.M., Gut, H., Grosshans, H., et al. (2018). Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition. Nature communications 9, 1549.
Tocchini, C., Keusch, J.J., Miller, S.B., Finger, S., Gut, H., Stadler, M.B., and Ciosk, R. (2014). The TRIM-NHL protein LIN-41 controls the onset of developmental plasticity in Caenorhabditis elegans. PLoS Genet 10, e1004533.