Pseudouridine modifications may regulate structure and protein recognition in mitochondrial mRNAs

Abstract

Pseudouridine(Ѱ) is one of the most abundant modifications in RNA that plays a crucial role in regulating mRNA function and metabolism. However, the exact mechanism by which Ѱ alters the structure, function and interaction of RBP with the mitochondrial mRNAs is still poorly understood. We investigated mitochondrial mRNAs with Ѱ modification using computational modelling and experimental validation. Predicted secondary structure revealed position-specific thermodynamic contribution. Two of the 4 mRNAs that had Ѱ at the stem position showed maximum destabilisation (0.9 and 1.2kcal/mol), whereas the Ѱ at the loop showed negligible energetic impact. Structural characterisation revealed altered base pair geometry, including twist, shear, torsion, and opening. Removal of pseudouridylation by incellulo knockdown of pseudouridine synthase enzymes resulted in a significant reduction of those mRNA levels in cells. To investigate the functional impact of Ѱ, we obtained a list of RBPs binding those regions from an experimentally validated database and examined whether Ѱ affects the interaction with RBPs. To address this, we performed molecular docking and MDS. We observed that Ѱ showed a differential effect with different RBPs. We found an additional hydrogen bond in the cases of DHX30, MTPAP, and TBRG4. We also observed an alteration in the hydrophobic interaction and the salt bridge. Molecular dynamics simulation confirmed altered complex stability, structural flexibility patterns, and reorganised hydrogen bonding networks in pseudouridine-containing complexes. Overall, this finding suggests that Ѱ modification in mitochondrial respiratory mRNAs might alter structure, thermodynamics and interaction with RBPs and may regulate the respiratory function in mitochondria.