PPP2R1A neurodevelopmental disorder is associated with congenital heart defects.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that regulates numerous biological processes. PPP2R1A encodes the scaffolding "Aα" subunit of PP2A. To date, nearly 40 patients have been previously reported with 19 different pathogenic PPP2R1A variants, with phenotypes including intellectual disability, developmental delay, epilepsy, infant agenesis/dysgenesis of the corpus callosum, and dysmorphic features. Apart from a single case, severe congenital heart defects (CHD) have not been described. We report four new unrelated individuals with pathogenic heterozygous PPP2R1A variants and CHD and model the crystal structure of several variants to investigate mechanisms of phenotype disparity. Individuals 1 and 2 have a previously described variant (c.548G>A, p.R183Q) and similar phenotypes with severe ventriculomegaly, agenesis/dysgenesis of the corpus callosum, and severe CHD. Individual 3 also has a recurrent variant (c.544C>T, p.R182W) and presented with agenesis of corpus callosum, ventriculomegaly, mild pulmonic stenosis, and small patent foramen ovale. Individual 4 has a novel variant (c.536C>A, p.P179H), ventriculomegaly, and atrial septal defect. To conclude, we propose expansion of the phenotype of PPP2R1A neurodevelopmental disorder to include CHD. Further, the R183Q variant has now been described in three individuals, all with severe neurologic abnormalities, severe CHD, and early death suggesting that this variant may be particularly deleterious.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant.

We report the identification and use of a mutant of the purine selective ribonuclease RNase U2 that randomly cleaves RNA in a manner that is directly compatible with RNA modification mapping by mass spectrometry. A number of RNase U2 mutants were generated using site-saturation mutagenesis. The enzyme activity and specificity were tested using oligonucleotide substrates, which revealed an RNase U2 E49A mutant with limited specificity and a tendency to undercut RNA. Using this mutant, RNA digestion conditions were optimized to yield long, overlapping digestion products, which improve sequence coverage in RNA modification mapping experiments. The analytical utility of this mutant was demonstrated by liquid chromatography tandem mass spectrometry (LC-MS/MS) mapping of several modified RNAs where 100% sequence coverage could be obtained using only a single enzymatic digestion. This new mutant facilitates more accurate and efficient RNA modification mapping than traditional highly base-specific RNases that are currently used.

The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA.

Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, its impact on RNA and RNA modifications is less studied. By using Escherichia coli tRNA (tRNA) as a model system, we identify the UVA (370 nm) susceptible chemical groups and bonds in a large variety of modified nucleosides. We use liquid chromatography tandem mass spectrometry to identify specific nucleoside photoproducts under in vitro and in vivo conditions, which were then verified by employing stable-isotope labeled tRNAs. These studies suggest that the -amino or -oxy groups of modified nucleosides, in addition to sulfur, are labile in the oxidative environment generated by UVA exposure. Further, these studies document a range of RNA photoproducts and post-transcriptional modifications that arise because of UVR-induced cellular stress.