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An altered basis of tRNA processing is linked to a distinct La protein in T. thermophila

ABSTRACT: La proteins bind the UUU-3’OH sequence found at the 3’-end of nascent RNA polymerase III transcripts, including pre-tRNAs, using a tandem arrangement of a winged-helix fold La motif (LaM) and an adjacent RNA recognition motif (RRM). La binding in diverse species results in protection of the 3’-end from exonucleases, stabilization of the pre-tRNA species and promotion of pre-tRNA folding until the UUU-3’OH containing trailer is endonucleolytically cleaved. Based on sequence conservation in the LaM, the Tetrahymena thermophila protein MLP1 (and related factors in other alveolates) has been hypothesized to function as a genuine La protein, despite the absence of the adjacent RRM found in La proteins of all prior investigated species. We show that MLP1 functions as a genuine La protein in that it binds and affects the processing of pre-tRNAs in T. thermophila and when expressed as a heterologous factor in fission yeast, however, unlike in other examined eukaryotes, depletion of MLP1 results in 3’-trailer stabilization. We also observed that 3’-trailers in T. thermophila are uniquely shortened relative to other examined eukaryotes, and that 5’-leaders in this species have evolved to disfavour pre-tRNA leader/trailer pairing. Our data suggest that the variant MLP1 architecture relative to other genuine La proteins is linked to an altered mechanism of tRNA processing in T. thermophila.

ORGANISM(S): Tetrahymena thermophila

PROVIDER: GSE199642 | GEO | 2022/09/13

REPOSITORIES: GEO

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Project description:We investigated how the biologically active form of vitamin D (1,25(OH)2D3) inhibits the epithelial-to-mesenchymal transition (EMT), a critical step in the development of a migratory phenotype in epithelial tumor cells, as well as migration in general, using osteosarcoma (OS) as a model. We looked at how 1,25(OH)2D3 affected pre- and post-EMT migratory states in non-metastatic MG63 and metastatic LM7 OS cell models. In scratch and Boyden chamber assays, 1,25(OH)2D3 inhibited cell migration and invasion, but LM7 cells responded significantly less than MG63 cells (50 vs 26 percent decrease, respectively). 1,25(OH)2D3 inhibited the expression of EMT-inducing genes (e.g., SNAI2) and extracellular contact regulatory genes in MG63 cells but not in LM7 cells (e.g., CD44, MMP3). In comparison to MG63 cells, LM7 cells had significantly lower baseline VDR transcript levels but higher SNAI2 transcript levels. Furthermore, ATAC-seq analysis revealed that 1,25(OH)2D3 epigenetically repressed SNAI2 in MG63 cells, implying that LM7 cells may be constitutively induced to undergo EMT via VDR de-repression mediated by increased SNAI2 levels. Both cell lines appeared to activate an antioxidative pathway involving SOD2 upregulation and concurrent reactive oxygen species (ROS) reduction (e.g., H2O2). ATAC-seq analysis revealed that 1,25(OH)2D3 does not epigenetically regulate SOD2 in MG63 cells, implying a previously unknown indirect link promoting anti-EMT and anti-metastatic oxidation states in both OS cell lines. We studied the fate and migration of mouse hair follicle stem cell progenitor cells after wounding with or without the Vdr because cutaneous wounding and metastasis involve similar signaling factors that regulate cell migration. Conditional loss of Vdr in epithelial progenitor cells promoted cell migration and wound healing in comparison to control animals, indicating that Vdr signaling inhibits cell migration in general. Our findings show that vitamin D signaling inhibits cell migration both in vivo and in vitro, and that inhibiting reactive oxygen species (ROS) in both pre-EMT and post-EMT OS cells is a possible vitamin D mechanism and therapeutic strategy for preventing the onset and progression of OS. MG63 human osteosarcoma cell line treated with vehicle (ethanol) or 1,25(OH)D (10nM) for 24 and 48 hours.

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An altered basis of tRNA processing is linked to a distinct La protein in T. thermophila

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