Effect of Aβ on RNase P activity.

<p>(<b>A</b>) A 5′-labeled (mt)tRNA^His precursor was cleaved with mtRNase P reconstituted from its recombinant components (TRMT10C-SDR5C1 and PRORP) in the presence of the indicated concentrations of freshly dissolved Aβ or scrambled sequence peptide. Reaction aliquots were withdrawn after 10, 30 and 60 minutes, stopped, and analyzed by denaturing PAGE and phosphor imaging. No enzyme was added to the mock reaction incubated for 60 minutes. The full length (mt)tRNA^His precursor and the released 5′ leader fragment are indicated on the right. (<b>B</b>) In experiments like that shown in (A), but with both, fresh and “<i>in vitro</i> aged”, oligomeric (oligo) peptide preparations, product formation was quantitatively analyzed and results plotted. Data are expressed relative to control reactions, to which only the solvent had been added. Mean and SD of three to five independent experiments are shown. (<b>C</b>) mtRNase P activity was assayed in the presence of freshly dissolved Aβ_1–28 and Aβ_25–35 fragments, and the results of three independent experiments were analyzed and plotted like in (B). (<b>D</b>) The RNase P activity of <i>A. thaliana</i> PRORP2 was assayed in the presence of different concentrations of fresh Aβ or scrambled sequence peptide and the results of four or five independent experiments were analyzed and plotted like in (B).</p

Effect of Aβ on the dehydrogenase activity of SDR5C1.

<p>L-3-hydroxyacyl-CoA dehydrogenase activity of SDR5C1 was measured with acetoacetyl-CoA as substrate and in presence of either (<b>A</b>) freshly dissolved or (<b>B</b>) “<i>in vitro</i> aged”, oligomeric (oligo) preparations of Aβ₄₂ or scrambled sequence peptide (scr). Data are expressed relative to the activity of control reactions, to which only the solvent had been added. Mean and SD of two (A) and three (B) independent experiments are shown.</p

Characterization of Aβ₄₂ preparations.

<p>Aβ₄₂ and scrambled sequence peptide (scr) preparations were analyzed by 10–16% Tris·Tricine-SDS-PAGE and silver staining. 75 pmols each of freshly dissolved, mostly monomeric (mono) or “<i>in vitro</i> aged”, oligomeric (oligo) peptide were loaded. The molecular weight of a reference protein ladder is shown on the left.</p

Effect of Aβ on tRNA:m¹R9 methyltransferase activity.

<p>(<b>A</b>) (mt)tRNA^Ile specifically labeled at position 9 was incubated with the TRMT10C-SDR5C1 complex in the presence of a methyl group donor and the indicated concentrations fresh Aβ₄₂ or scrambled sequence peptide. No enzyme was added to the mock reaction. The tRNA hydrolysate was resolved by TLC and visualized by phosphor imaging. The 30-minute time-point of the reactions is shown. (<b>B</b>) Product formation in experiments like that shown in (A) was quantitatively analyzed and results plotted. Data are expressed relative to control reactions, to which only the solvent had been added. Mean and SD of three or four independent experiments are shown. (<b>C</b>) The methyltransferase activity of yeast Trm10p was assayed in the presence of different concentrations of fresh Aβ or scrambled sequence peptide and the results of four independent experiments were analyzed and plotted like in (B).</p

Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs.

Stress-induced tRNA fragmentation upon environmental insult is a conserved cellular process catalysed by endonucleolytic activities targeting mature tRNAs. The resulting tRNA-derived small RNAs (tsRNAs) have been implicated in various biological processes that impact cell-to-cell signalling, cell survival as well as gene expression regulation during embryonic development. However, how endonuclease-targeted tRNAs give rise to individual and potentially biologically active tsRNAs remains poorly understood. Here, we report on the in vivo identification of proteins associated with stress-induced tsRNAs-containing protein complexes, which, together with a 'tracer tRNA' assay, were used to uncover enzymatic activities that can bind and process specific endonuclease-targeted tRNAs in vitro. Among those, we identified conserved ATP-dependent RNA helicases which can robustly separate tRNAs with endonuclease-mediated 'nicks' in their anticodon loops. These findings shed light on the existence of cellular pathways dedicated to producing individual tsRNAs after stress-induced tRNA hydrolysis, which adds to our understanding as to how tRNA fragmentation and the resulting tsRNAs might exert physiological impact

A novel <i>HSD17B10</i> mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

<p>We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene <i>HSD17B10</i> for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in <i>HSD17B10</i> cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5′-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.</p