Poster abstracts
Poster number 16 submitted by Kaylee Grabarkewitz
Study of the conformational diversity of a tRNAArg variant expressed in individuals with dextrocardia
Kaylee Grabarkewitz (The Department of Chemistry and Biochemistry; Center for RNA Biology; Resource for Native MS Guided Structural Biology, The Ohio State University, Columbus, OH), Moulisubhro Datta (Center for RNA Biology; Molecular Cellular and Developmental Biology Program, The Ohio State University, Columbus, OH), Venkat Gopalan (The Department of Chemistry and Biochemistry; Center for RNA Biology, The Ohio State University, Columbus, OH), Vicki Wysocki (The Department of Chemistry and Biochemistry; Center for RNA Biology; Resource for Native MS Guided Structural Biology, The Ohio State University, Columbus, OH)
Abstract:
Dextrocardia with situs inversus is a rare congenital disease in which the vital organs within the chest and abdomen are positioned opposite to the typical arrangement (e.g., heart is not located in the left side of the thoracic cavity, but rather on the right). One possible cause of dextrocardia with situs inversus that we are investigating is a single-nucleotide polymorphism (SNP) in the 3' UTR of the HES7 gene, specifically focusing our attention on a tRNAArg UCU isodecoder (tRNA named TCT-2-1) encoded immediately downstream to HES7 and on the opposite strand. We hypothesize that the SNP, located in the trailer sequence, causes an extension of the acceptor stem of the precursor of TCT-2-1, a change that could potentially impair processing by RNase P, which cleaves the 5'-leader of precursor tRNAs (pre-tRNAs). We first mapped the native 5'-leaders and 3'-trailers in pre-TCT-2-1 and determined that while the wild-type (WT) and mutant (MT) both had a 4-nucleotide (nt) 5'-leader, their 3'-trailers varied, with the Mt having a wide range of longer trailers because the mutation disrupts a RNA Pol III termination site. Based on the distribution of trailer lengths, we synthesized invitro constructs with 4 nt leader and either 4 nt (WT) or 8 nt (MT) trailer (4L4T or 4L8T, respectively), and sought to map confirmational changes engendered by the point mutation. Native mass spectrometry studies confirmed the presence of exclusively monomeric species. Further analysis with ion mobility mass spectrometry (IM-MS) using the Waters Select Series Cyclic and Agilent 6560 IMS showed differences between the WT and MT pTCT-2-1. Collision-induced dissociation (CIU) studies of the tRNA species showed a collapse in structure as collisional voltages were increasing, but only the WT species had an expansion and unfolding feature at the high collisional voltages. For both the 4L4T and 4L8T in the absence of Mg2+ the Mut tRNA species had a higher CIU50 value than that of the WT, indicate a more sample structure. The nMS-IM data suggests differences in conformation and stability, indicated that the mutation in the trailer sequence of the tRNA allows for a more stable conformation to be adopted.
Keywords: tRNA, Ion Mobility Mass Spectrometry