The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules *

... consequence, the presence of pseudouridine generally rigidifies RNA secondary structures; thus it has been proposed to contribute toward the stabilization of a particular structural motif. Accordingly, pseudouridylation is found predominantly in tRNAs, rRNAs, and other small non-coding RNAs ( 7 ) for which proper structure is critical for function. In humans, 13 different pseudouridine synthases have been identified. Of these 13 enzymes, five have been shown (PUS1) or predicted (PUSL1, TRUB2, RPUSD3, and

... components based on the formation of distinct foci and co-localization with the established MRG component FASTKD2 ( 19 , 20 ) ( Fig. 1 B ). From this analysis, we identified 12 new MRG-associated proteins ( Table 1 ), including proteins involved in RNA processing (FASTKD1, PTCD1, and PTCD2), RNA modification (TFB1M, TRUB2, RPUSD3, and RPUSD4), mitoribosome assembly (ERAL1 and NOA1/C4orf14), and structural components of mitoribosome (MRPL47, MRPS7, and MRPS9). Interestingly, some of the novel MRG-associated proteins (ERAL1, PTCD1, RPUSD3, and TRUB2) also emerged to physically interact with the already known MRG protein components FASTK, FASTKD2, GRSF1, and MRPP1 in co-immunoprecipitation experiments ( supplemental Fig. S2 and supplemental Table S1 ). From these observations, we can confirm that the MRGs contain proteins participating in almost all aspects of mitochondrial gene expression. FIGURE 1. Identification of RNA-binding proteins associated with MRGs. A , schematic representation of the

..., mitochondrial RNA processing PTCD2 Pentatricopeptide repeat-containing protein 2, mitochondrial RNA processing RPUSD3 RNA pseudouridylate synthase domain-containing protein 3 RNA modification RPUSD4 RNA pseudouridylate synthase domain-containing protein 4 RNA modification TFB1M Dimethyladenosine transferase 1, mitochondrial RNA modification TRUB2 Probable tRNA pseudouridine synthase 2 RNA modification From the list of the proteins specifically associated with MRGs, we selected three poorly characterized proteins, TRUB2, RPUSD3, and RPUSD4, which belong to a group of five mitochondrial pseudouridine synthases (see the Introduction). To determine whether MRG localization is a general feature of this protein family, we investigated the submitochondrial localization of each of the five enzymes (TRUB2, RPUSD3, RPUSD4, PUS1, and PUSL1) by immunofluorescence. For this, each of these enzymes was tagged with a FLAG peptide at the C terminus and expressed at a low level in 143B cells. We observed a clear enrichment of RPUSD3, RPUSD4, and TRUB2 in MRGs, whereas neither PUS1 nor PUSL1 appeared to be specifically enriched in discrete foci ( Fig. 2 A ). We conclude therefore that the submitochondrial localization of the pseudouridine synthases is a specific property of each member of the family. FIGURE 2. Submitochondrial localization of the mitochondrial pseudouridine synthases. A , confocal analysis of each of the five mitochondrial pseudouridine synthases. 143B cells were transfected with expression

... conclusion that multiple steps in mitochondrial gene expression take place within this compact submitochondrial compartment ( 15 ). The newly identified proteins are implicated in mtRNA processing (FASTKD1, PTCD1, and PTCD2), mtRNA modification (TFB1M, TRUB2, RPUSD3, and RPUSD4), mitoribosome assembly (ERAL1 and NOA1/C4orf14), and mitoribosome structure (MRPL47, MRPS7, and MRPS9). This expands the previously established list of MRG-associated proteins that includes GRSF1 ( 23 , 24 ), FASTK ( 20

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Gene: TRUB2 - TruB pseudouridine (psi) synthase family member 2 - (NCBI)

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