Poster abstracts

Poster number 40 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), Ila A. Marathe (Chemistry and Biochemistry, The Ohio State University), Lauren Levesque (Department of Molecular Genetics, The Ohio State University), Vicki H. Wysocki (Chemistry and Biochemistry, The Ohio State University), 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 and situs inversus2. This SNP lies in the 3' UTR of the Notch signaling gene HES 7. However, mutation of HES 7 is associated with skeletal and not laterality defects3,4. Interestingly, we noted that a tRNAArg UCU (n-Tr25) is encoded on the opposite strand from HES 7 and in the region that contains the SNP. Because several genes that dictate left-right symmetry have large numbers of AGA codons 5, we hypothesized that alterations to n-Tr25 levels from impaired 5' maturation (of the SNP-bearing mutant) by RNase P could lead to laterality defects.
Native PAGE experiments revealed that while the wildtype pre–n-Tr25 exhibited a Mg2+-dependent shift to folded states, the mutant appeared diffuse, reflective of structural heterogeneity. Native mass spectrometry and mass photometry confirmed that both RNAs existed as a monomer under the conditions tested. 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. This is consistent with previous studies that showed RNase P prefers a single-stranded region preceding 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). Preliminary data indicate that that the SNP leads to pre–n-Tr25 transcripts with longer 3' trailers presumably due to altered pol III termination. We are excited to add to the growing evidence that point mutations in tRNAs affect their structure and maturation to culminate 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