Nikhil Sunny (IISER Pune, India)
LinkedIn: @Nikhil Sunny; X: @Nikhil__Sunny__
Abstract: HuD is an RNA-binding protein (RBP) essential for neuronal development and glucose homeostasis. It has tandemly arranged three RNA recognition motifs (RRMs). Multiple isoforms, namely, HuD A, HuD B, and HuD D—have been reported that are differentially expressed in various tissues. The A and B isoforms both feature an unstructured N-terminal region; however, the A isoform has five additional amino acids when compared to the B isoform. This difference significantly impacts the RNA-binding and translation of insulin 2 mRNA. While the structure of HuD RRM12 is known, it does not include the unstructured N-terminal region, creating a gap in understanding its role in RNA targeting. In this study, we focus on understanding the role of the unstructured N-terminal region of A and B isoforms in the RNA-binding activity of the RRM1 domain by using NMR-based dynamics experiments and other biophysical techniques. FOur preliminary results show that the presence of the N-terminal leads to line-broadening and the disappearance of peaks in the 2D 15N-1H HSQC spectra. The disappeared peaks are mainly from the N-terminal region and the possible site of intra- and/or intermolecular interactions. In the presence of the N-terminal region, CSP is found in or near the RNP motifs of RRM1, which are the sites for RNA binding. We believe that this study will provide insights into how intrinsically disordered regions affect the intrinsic dynamics and RNA-binding activity of the RRM.
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Interesting!
Although I believe you are planning on further experiments to confirm the interaction of the N-ter with the folded domain, can you comment on the effect it could have on the binding to RNA?
Also, how do the R1 and R2 of the N-ter tail evolve as a function of residue number?-
1) The RNA-binding region can either be masked, reducing overall binding affinity, or it can function as an auxiliary region that enhances binding affinity. Since this RNA recognition motif (RRM) is a weak binder, we are working on optimizing the experimental parameters for the binding studies. I suspect it will act as an auxiliary region facilitating RNA binding, as shown from EMSA studies on Isoforms of HuD (full-length)(https://doi.org/10.1371/journal.pone.0194482)
2) “R1 and R2 of the N-ter tail evolve as a function of residue number.” I haven’t done those experiments. That’s the next part of my project.
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Interesting work:
What type of experiments you are planning to look for exact functional role of this protein?
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I will be doing in vitro studies only; majorly binding studies with RNA using ITC, NMR, fluorescence, etc.
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