Journal: International Journal of Molecular Sciences

Article Title: Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae

doi: 10.3390/ijms22147298

Figure Lengend Snippet: RBS1 and PRT1 as overdose suppressors of the Pol III assembly mutant rpc128-1007 . ( A ) Expression of the GCN4-lacZ in rpc128-1007 mutant. The rpc128-1007 mutant and isogenic wild-type strains, transformed with p180 plasmid ( GCN4-lacZ , CEN , URA3 ), were grown under nonstarvation or starvation conditions, as described in the Materials and Methods section. Extracts were prepared and assayed for β-galactosidase activity (expressed as nanomoles of o -nitrophenol-β-D-galactopyranoside hydrolyzed per minute per microgram of total protein). The reported values are averages of three independent measurements (number of biological replicates n = 3). ( B – D ) The rpc128-1007 mutant was transformed with a control empty vector [–] or multicopy plasmids [ PRT1 ], [ RBS1 ], and [ RBS1 -R3H]. ( B ) The effect of suppressors on GCN4-lacZ expression. Cells harboring p180 plasmid ( GCN4-lacZ , CEN , URA3 ) were grown in YPD, harvested in log phase. β-galactosidase activity was determined and calculated relative to the amounts in the wt strain, which was set as 1 ( n = 3). ( C ) Suppression of the rpc128-1007 growth phenotype. Cells that were grown on a YPD plate were replicated on YPD plates and incubated for three days at the respective temperatures. ( D ) Determination of total tRNA levels. Small RNA species were isolated and separated on a 7 M urea–6% polyacrylamide gel using equal amounts of RNA per lane (2.5 μg) and stained with ethidium bromide ( n = 3). (E) Determination of specific tRNA levels by northern hybridization with probes specific for mature tRNA i Met , tRNA Phe , and tRNA Tyr . tRNA amounts in D and E were normalized to the loading control (5.8 S rRNA) and calculated relative to amounts in the wt strain, which was set as 1. Bars represent the mean ± standard deviation (SD) of three independent experiments ( n = 3). Values of p were calculated using a two-tailed paired t -test: * p < 0.05; ** p < 0.01; *** p < 0.001. Asterisks just under bars show the p -value in comparison to the level in wt; other comparisons are annotated by lines. To clarify the plots, bars are colored as follows: rpc128-1007 with control vector—pink; with [ PRT1 ], [ RBS1 ], and [ RBS1 -R3H]—yellow, dark blue, and light blue, respectively; grey was used for control strains. The same color code and calculation of p -values are used in the figures hereafter.

Article Snippet: Total RNA was extracted from yeast cells using the Bead-beat Total RNA Mini kit (A&A Biotechnology, Gdańsk, Poland, catalogue no. 031-25BB) and purified using Clean-Up RNA Concentrator (A&A Biotechnology, catalogue no. 039-25C).

Techniques: Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation, Activity Assay, Incubation, Isolation, Staining, Northern Blot, Hybridization, Standard Deviation, Two Tailed Test