Poster abstracts

Poster number 68 submitted by Moulisubhro Datta

Exploring the role of a tRNA SNP in the molecular basis of dextrocardia, a laterality disorder

Moulisubhro Datta (MCDB, The Ohio State University), Kaylee Grabarkewitz (Chemistry and Biochemistry, The Ohio State University), Vicki H. Wysocki (Chemistry and Biochemistry, Georgia Institute of Technology ), Susan E. Cole (Department of Molecular Genetics, The Ohio State University), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University)

Abstract:
Dextrocardia is a rare congenital condition in which the heart is misaligned in the thoracic cavity.1 A recent case study identified homozygosity for a rare SNP allele (chr 17) as a possible cause for dextrocardia.2 This SNP lies in the 3' UTR of HES7, a crucial Notch signaling gene involved in skeletal but not laterality defects.3,4 Interestingly, we noted that a tRNAArgUCU (TCT2-1) is encoded on the opposite strand and in the same region carrying the SNP. Because several genes that dictate left-right symmetry have large numbers of AGA codons,5 we hypothesized that SNP induced alterations to TCT2-1 levels due to impaired 5' maturation by RNase P culminates in laterality defects. Northern blot experiments showed the presence of longer precursor (pre)-tRNA transcripts in the mutant (MUT) compared to the wild type (WT), due to the disruption of an RNA Pol III termination signal by the SNP. Sequence mapping revealed that both the WT and MUT pre-TCT2-1 carry a 4-nucleotide (nt) leader but while the WT pre-TCT2-1 has a 4-nt trailer (termed 4L4T) the MUT pre-TCT2-1 has an 8- nt trailer (termed 4L8T). Native PAGE experiments with in vitro transcripts highlighted a Mg2+-dependent alteration in conformation. Collision-induced dissociation studies revealed that, in the presence of Mg2+, the MUT is more stable requiring a higher energy (221 V) for the structure to be perturbed compared to the WT (211 V). The MUT also has a larger collisional cross section than the WT. We also found that in vitro reconstituted Escherichia coli RNase P cleaves the mutant (with a presumed longer acceptor–T-stem stack) at a slower rate, consistent with previous studies which showed that RNase P prefers a single-stranded region to precede the pre-tRNA cleavage site.6-8 We found that the laterality disorder-associated tRNA SNP dampens the rate of processing by RNase P but recognize the need to assess functional levels of this tRNA in vivo; reporter constructs in cultured cells and transgenic mice will better inform this scenario (ongoing studies). We are excited to add to the growing evidence that point mutations in tRNAs affect their structure and maturation to end in disease and highlight the unexpected ripple effects of SNPs.

References:
1. Deng et al. (2015) Expert Rev. Mol. Med. 16: e19.
2. Netravathi et al. (2015) BMC Med. Gen. 16: 1-8.
3. Sparrow et al. (2010) Eur. J. Hum. Genet. 18: 674-679.
4. Luo et al. (2016) Sci. Rep. 6: 31583.
5. Orellana et al. (2021) Mol Cell 81: 3323-3338.
6. Kirsebom & Svärd (1992) Nucleic Acid Res. 20: 425-432.
7. Lee et al. (1997) RNA 3: 175-185.
8. Torabi et al. (2021) RNA 27: 1140-1147.

Keywords: tRNA, mutation, disease