Lnc-EPB41-Protein Interactions Associated with Congenital Pouch Colon

Abstract

Congenital Pouch Colon (CPC) is a rare anorectal anomaly common to northwestern India, specifically Rajasthan. Despite efforts to understand the clinical genetic makeup of CPC, no attempt on identifying non-coding RNAs was done. We have earlier reported CPC's rare variants from whole exome sequencing (WES) across 18 affected samples in a total of 64 subjects. A Smith⁻Waterman algorithm was used to infer a couple of lncRNAs from WES samples of CPC with predictions from the Noncode database. Further screening and quantification using polymerase chain reaction (PCR), we ascertained interactions using Micro Scale Thermophoresis (MST). We report the role of lnc-EPB41-1-1 shown to be promiscuously interacting with KIF13A substantiating their role in regulation.

Keywords: congenital pouch colon; long non coding RNA; microscale thermophoresis; protein interaction; whole exome sequencing.

Figure 1

(a) RNAfold prediction showing base-pairing probabilities of the secondary structures of lncRNA wherein the centroid secondary structure (right)is shown to have a minimum free energy(MFE) of −322.60 kcal/mol indicating that the structure is in thermodynamic ensemble; (b) plausible role of lncRNA towards regulation with lnc-EPB41-1-1 known to be associated with open chromatin elements (OCE), promoters and enhancers; (c) lnc-EPB41-1-1 potentially shown to be interacting with KIF13A with the red edge indicating the predictions from lncPro and RNA Protein Interaction predictor (RPI-Pred).

Figure 2

(a) the binding of fluorescently labelled protein to affected lnc-EPB41-1-1 is analysed with microscale thermophoresis (MST). RNA was titrated and change in the thermophoretic signal leads to binding affinity of 30 nM ± 11.6 nM with S/N: 10; (b) the interaction of labelled protein with wild type lnc-EPB41-1-1 is measured and MST showed no/weak binding under the tested conditions.

Figure 3

The pathway network map of KIF13A with pink edges (showing physical interactions), purple (co-expression) and violet (co-localization) and the blue thick edges showing the pathways.

Figure 4

Flow chart demonstrating the methodology to characterize the lncRNA-protein interactions.