Journal: Nature Communications

Article Title: A tRNA half modulates translation as stress response in Trypanosoma brucei

doi: 10.1038/s41467-018-07949-6

Figure Lengend Snippet: Depletion of endogenous tRNA Thr 3′ halves alleviates the stimulatory effects during translation. a Scheme of the strategy used for affinity purification of the endogenous tRNA Thr 3′ half from the pool of small RNAs (size range between 30–40 nucleotides). As a control the same procedure was performed in the absence of a biotinylated antisense oligonucleotide (ASO). b Northern blot analysis of RNA extracted from the samples obtained after affinity purification showing the successful isolation of the tRNA Thr 3′ half (see also Supplementary Figure ) when using the biotinylated ASO (fraction II+) and its partial depletion from the corresponding flow through (compare fractions III, − and +ASO). A representative blot of in total two independent affinity purification experiments is shown. c Depletion of endogenous tRNA Thr 3′ halves from the pool of small RNAs results in reduced in vitro translation activities. The effects of RNA fractions obtained after affinity purification were tested on in vitro translation. Quantification (mean and standard deviation) shows the average of three independent experiments. d Chemically modified ASO complementary to the tRNA Thr 3′ half were electroporated into T. brucei cells and their effect on in vivo translation was investigated by metabolic labeling during stress recovery. The autoradiograph shows a representative SDS polyacrylamide gel in which metabolic labeling was performed in cells electroporated without an ASO (−) or cells electroporated with one (a) or two (a+b) different ASO targeting endogenous tRNA Thr 3′ halves. Coomassie staining of the gel (bottom panel) serve as loading control. Quantification of three independent metabolic labeling experiments is shown on the right. As specificity control metabolic labeling was performed also after electroporation of an analogous ASO without any sequence complementarity to the tRNA Thr 3′ half (ctr). Significance according to paired Student’s t -test: ** P ≤ 0.01

Article Snippet: The mixture was electroporated twice using a Bio-Rad gene pulser II (1.2 kV, 25 μF, and 0 Ohm) in a 4 mm electroporation cuvette (EP-104, Cell Projects Ltd .).

Techniques: Affinity Purification, Control, Northern Blot, Isolation, In Vitro, Standard Deviation, Modification, In Vivo, Labeling, Autoradiography, Staining, Electroporation, Sequencing