Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis.

Posttranslational protein arginylation catalyzed by arginyl transferases is a mechanism to regulate multiple physiological processes. This protein arginylation reaction uses a charged Arg-tRNAArg as the donor of arginine (Arg). The inherent instability of the ester linkage of the arginyl group to the tRNA, which is sensitive to hydrolysis at the physiological pH, makes it difficult to obtain structural information on how the arginyl transfer reaction is catalyzed. Here, we describe a methodology to synthesize stably charged Arg-tRNAArg that would facilitate structural analysis. In the stably charged Arg-tRNAArg, the ester linkage is replaced with an amide linkage, which is resistant to hydrolysis even at alkaline pH.

tRNA methylation resolves codon usage bias at the limit of cell viability.

Codon usage of each genome is closely correlated with the abundance of tRNA isoacceptors. How codon usage bias is resolved by tRNA post-transcriptional modifications is largely unknown. Here we demonstrate that the N1-methylation of guanosine at position 37 (m1G37) on the 3'-side of the anticodon, while not directly responsible for reading of codons, is a neutralizer that resolves differential decoding of proline codons. A genome-wide suppressor screen of a non-viable Escherichia coli strain, lacking m1G37, identifies proS suppressor mutations, indicating a coupling of methylation with tRNA prolyl-aminoacylation that sets the limit of cell viability. Using these suppressors, where prolyl-aminoacylation is decoupled from tRNA methylation, we show that m1G37 neutralizes differential translation of proline codons by the major isoacceptor. Lack of m1G37 inactivates this neutralization and exposes the need for a minor isoacceptor for cell viability. This work has medical implications for bacterial species that exclusively use the major isoacceptor for survival.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9.

We present the complete chemical structures of the rRNAs from the eukaryotic model organism, Saccharomyces cerevisiae The final structures, as determined with mass spectrometry-based methodology that includes a stable isotope-labelled, non-modified reference RNA, contain 112 sites with 12 different post-transcriptional modifications, including a previously unidentified pseudouridine at position 2345 in 25S rRNA. Quantitative mass spectrometry-based stoichiometric analysis of the different modifications at each site indicated that 94 sites were almost fully modified, whereas the remaining 18 sites were modified to a lesser extent. Superimposed three-dimensional modification maps for S. cerevisiae and Schizosaccharomyces pombe rRNAs confirmed that most of the modified nucleotides are located in functionally important interior regions of the ribosomes. We identified snR9 as the snoRNA responsible for pseudouridylation of U2345 and showed that this pseudouridylation occurs co-transcriptionally and competitively with 2'-O-methylation of U2345. This study ends the uncertainty concerning whether all modified nucleotides in S. cerevisiae rRNAs have been identified and provides a resource for future structural, functional and biogenesis studies of the eukaryotic ribosome.

The C-terminal cytoplasmic tail of hedgehog receptor Patched1 is a platform for E3 ubiquitin ligase complexes.

The Sonic hedgehog (Shh) signaling pathway plays a crucial role in cell proliferation and differentiation via Patched1 (Ptc1), a 12-pass transmembrane receptor protein. The C-terminal cytoplasmic tail of Ptc1 can be cleaved to release the 7th intracellular domain (ICD7), whose function is still unclear. In this study, we found that the ICD7 fragment of Ptc1 associates with polyubiquitinated species. Using mass spectrometry, we identified a cluster of E3 ubiquitin ligase complex as novel Ptc1 ICD7-binding proteins. In particular, Ptc1 ICD7 interacted with most components of the Cullin-2 (CUL2)-based E3 ligase complex, including TCEB1 (EloC), TCEB2 (EloB), ZYG11B, and CUL2 itself. To address the significance of CUL2-based E3 ligase in Ptc1 function, we examined the effects of CUL2 knockdown on Shh-induced osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Indeed, knockdown of CUL2 abolished the Shh-induced stem cell differentiation. These results suggest that CUL2-based E3 ligase complex may play a role in Shh- and Ptc1-dependent signaling pathways.