Fig. 4: Mlp1 promotes tRNA-mediated suppression in the absence of protection of 3’-trailers.
From: Altered tRNA processing is linked to a distinct and unusual La protein in Tetrahymena thermophila
a tRNA-mediated suppression assay of Schizosaccharomyces pombe ySH9 strain transformed with pRep4 encoded Sla1p (positive control; S. pombe La protein), Mlp1 or indicated Mlp1 mutants (n = 3 biologically independent samples). Protein expression levels are shown by western blot in Supplementary Fig. S6a. Levels of suppressor tRNA stabilization is based on the color of the colonies and annotated as follows: full suppression (+++), near full suppression (++), intermediate suppression (+), no suppression (−). Top: diagram showing domain architecture of Mlp1. b Northern blot to determine 3’-end protection of suppressor pre-tRNA-SerUCA in ySH9 transformants shown in (a) (n = 2 biologically independent samples). Accumulation of suppressor pre-tRNA-SerUCA was determined using an intron complementary probe and mature tRNA using a tRNA body probe leading to the detection of both pre-tRNA and mature tRNA. The asterisk indicates a 3’-processed pre-tRNA intermediate. An excess unlabeled probe complementary to endogenous SerUGA was added to avoid cross-reaction between suppressor tRNA and endogenous tRNA. U5: loading control. c Northern blot to determine 3’-end protection of endogenous pre-tRNA LysCUU in ySH9 transformants shown in (a), with suppressor tRNA stabilization levels (see panel a) shown above the blot (n = 3 biologically independent samples). Accumulation of pre-tRNA LysCUU intermediates was determined using an intron complementary probe which detects unprocessed 5’-leader and 3’-trailer-containing pre-tRNA (band a), 5’-leader processed pre-tRNA (band b) and both 5’-leader and 3’-trailer processed pre-tRNA (band c) in Sla1p transformants (positive control; lane 2). The 3’-processed, 5’-leader containing pre-tRNA intermediate is annotated as band d. The same blot was probed with a 3’-trailer complementary probe, a 5’-leader complementary probe and a mature tRNA complementary probe. U5: loading control. d Sanger sequencing of clonal isolates corresponding to cDNAs derived from 3’-terminal sequences from Sla1p- and Mlp1-immunoprecipitated pre-tRNAs in ySH9 as transformed in (a–c) (n = 2 biologically independent samples). Western blot of Sla1p- and Mlp1-immunoprecipitations shown in Supplementary Fig. S6b. Source data are provided as a Source Data file.
