PMC:4573257 / 32214-33940 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4573257","sourcedb":"PMC","sourceid":"4573257","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4573257","text":"Hypomodification of G37 in Mitochondrial tRNA in Affected Individuals and upon Downregulation of TRMT5 Expression\n(A) A radioactively labeled, complementary primer (red) is annealed to mt-tRNALeu(CUN) and subjected to a RT-PEx reaction. The presence of m1G37 results in RT-PEx pausing, producing a shorter product (dark blue). However, in the absence of m1G37, the extension is able to progress until stalling due to the lack of a dNTP (dTTP in the above case, light blue), producing a longer product.\n(B) Separation and detection of products of RT-PEx reactions preformed on RNA extracted from skin fibroblasts (FB) of healthy controls (C1 and C2) and the affected individuals (65205 and 73901; left panel). The gel shows a representative result of at least three biological replicates (right panel). Quantification values represent percentage of RT-PEx reaction readthrough the G37 site as the average of the dTTP-induced stalling intensity to the total stalling (dTTP-induced stalling + m1G37-induced stalling). Error bars = 1 SEM; n = 5, ∗p \u003c 0.05, ∗∗∗p \u003c 0.001, unpaired two-tailed Student’s t test with C1.\n(C) RT-PEx experiment performed as in (B) on RNA extracted from skeletal muscle (SKM) of healthy control individuals (C1 and C2) and affected individuals (65205 and 73901). Error bars = 1 SEM; n = 3, ∗∗∗p \u003c 0.001, unpaired two-tailed Student’s t test with C1.\n(D) Separation and detection of RT-PEx reactions on HeLa cells-derived RNA following a 6-day siRNA-mediated depletion of TRMT5. Two different siRNA were used (HSS126475 and HSS126476, Life Technologies). Untransfected cells and cells transfected with siRNA against GFP (Dsc-1001, Lonza) were used as controls. Quantification values displayed as per (B).","divisions":[{"label":"p","span":{"begin":0,"end":113}},{"label":"p","span":{"begin":114,"end":501}},{"label":"p","span":{"begin":502,"end":1112}},{"label":"p","span":{"begin":1113,"end":1372}}],"tracks":[]}