Volume 18, Issue 6, Pages 711-722 (June 2005) Interdependence between Transcription and mRNP Processing and Export, and Its Impact on Genetic Stability  Rosa Luna, Sonia Jimeno, Mercedes Marín, Pablo Huertas, María García-Rubio, Andrés Aguilera  Molecular Cell  Volume 18, Issue 6, Pages 711-722 (June 2005) DOI: 10.1016/j.molcel.2005.05.001 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Gene Expression Analyses of Mutants Affected in Different Steps of mRNP Biogenesis and Export (A) Northern analysis of lacZ, GAL1, and TEF1 mRNAs of mutants transformed with centromeric plasmids p416GAL1lacZ or p416TEF1lacZ carrying lacZ fused to the GAL1 or TEF1 promoter. RNA was isolated from midlog phase cultures grown in synthetic complete medium without uracil and with 2% galactose or glucose, depending on whether transformants carried p416GAL1lacZ or p416TEF1lacZ, respectively. One representative experiment is shown for each mutant studied and its corresponding isogenic wt strain. (B) Quantification data of the lacZ mRNAs obtained with p416TEF1lacZ (mutants marked with asterisks or p416GAL1lacZ (no mark). The average and SD of three different transformants are represented. All mutants were analyzed together with their isogenic wt strains. Values are given with respect to the average wt value, normalized to 100%. The mutants have been grouped by their reporter function: I, nuclear cap binding complex; II, splicing; III, 3′ end processing and termination; IV, nuclear mRNA degradation; V, nuclear RNA associated proteins; VI, mRNA export; VII, protein nuclear export; VIII, translation; IX, cytoplasmic mRNA degradation; and X, others (see Table SI for details). Data on tho2Δ and sub2Δ have been published previously. (C) mRNA values of the endogenous GAL1 and TEF1 (*). The analysis of GAL1 and TEF1 expression was performed only in the transformants showing lacZ expression levels below 75% the wt value (gray boxes in [B]). Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Recombination Analyses of Mutants Affected in Different Steps of mRNP Biogenesis and Export Mutant strains studied were those showing lacZ mRNA accumulation levels below 75% of the wt (see Figure 1). Recombination was analyzed in the plasmidic recombination systems LY and L, carrying 600 bp leu2 repeats or in the chromosomal direct-repeat recombination system leu2-k::ADE2-URA3::leu2-K (Lk-AU) based on 2.16 kb repeats. A small diagram of each system used (not drawn to scale) is shown in which repeats are shown as gray boxes, and gray arrows indicate relevant transcripts produced by the constructs. For each genotype, the median and SD of three to four median frequencies obtained from an identical number of fluctuation tests performed with two different transformants are shown. All mutants were analyzed together with their isogenic wt strains. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Recombination Analysis of the rna14-1 and rrp6Δ Mutants The frequency of Leu+ recombination was determined in the plasmidic systems L-lacZ and GL-lacZ based on 600 bp leu2 repeats under the LEU2 or the GAL1 promoter, respectively. A diagram of the repeat systems used is shown with the repeats as gray boxes; arrows indicating the transcripts produced and the position of the promoter (Prm) and terminator (Ter) of transcription are shown. Recombination frequencies are represented according to the levels of transcription. LOW transcription refers to the GL-lacZ systems in strains cultured in 2% glucose, MEDIUM to L-lacZ in 2% glucose, and HIGH to GL-lacZ in 2% galactose. Other details are as in Figure 2. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of the Genetic and Functional Interaction of rna14-1 and tho2Δ (A) Inviability of rna14-1 tho2Δ mutants. Tetrad analysis of the diploid was constructed by crossing the strains tho2Δ and rna14-1. The white open squares indicate the position of tho2Δ rna14-1 double mutant spores, which did not form colonies (top). Capacity of tho2Δ and tho2Δ rna14-1 mutants transformed with the plasmid pRS316THO2, containing URA3 and THO2, to grow on SC-FOA and SC media. 10-fold serial dilution spots were plated (bottom). (B) Transcription readthrough analysis. Northern analysis of transcription of the tet::LEU2 fusion construct in rna14-1 and mutants of the THO complex. The diagram of the tet::LEU2 fusion construct showing the two termination sites (Ter) is shown on the top. The 32P-labeled internal 0.6 kb ClaI-EcoRV LEU2 fragment and the 589 bp 25S rDNA fragment obtained by PCR were used as probes. LEU2 transcripts are indicated depending on the termination site used. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 In Vitro Transcription Elongation of Wt, rna14-1, rna15-1, and rrp6Δ Strains (A) Scheme of the two G-less cassette system of plasmid pGCYC1-402 used for the analysis of in vitro transcription elongation. RNase T1 treatment of the mRNA driven from the GAL4-CYC1 promoter, which is activated by purified Gal4-VP16, renders two fragments corresponding to the G-less cassettes. (B) In vitro transcription assays of WCEs from wt, rna14-1, rna15-1, and rrp6Δ strains. Each reaction was stopped after 30 min, treated with RNaseT1, and run in a 6% PAGE. Transcription reactions were made at 23°C with WCEs obtained from cells grown at 30°C. Two bands from each G-less cassette were obtained, probably due to incomplete action of RNaseT1. Efficiency of transcription elongation was determined as the percentage of total transcripts that reached the 376 nt G-less cassette with respect to the transcripts that covered the 84 nt cassette. Radioactivity incorporated into the G-less cassettes was quantified in a Fuji FLA3000 and normalized with respect to the C content of each G-less cassette. The mean value and SD of three independent experiments are shown. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 ChIP Analysis of RNAPII at the lacZ Gene in rna14-1 and rrp6Δ Strains ChIP experiments were made with rna14-1, rrp6Δ, and isogenic wt strains transformed with p416TEF1lacZ or p416GAL1lacZ. A scheme of lacZ and PCR fragments analyzed are shown. One representative polyacrylamide-gel electrophoresis is shown for each genotype. The band marked by an asterisk is an internal background control from the nontranscribed region on chromosome V. The other bands correspond to the lacZ PCR-specific fragments. The relative abundance of each band with respect to the background is represented (see Experimental Procedures). For each strain, ChIPs were performed in two to three different transformants each, and PCRs were repeated at least three times for each transformant. The mean value and SD are shown. Prm indicates promoter and IP indicates immunoprecipitate. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 ChIP Analysis of Tho2 at the GAL1 and PMA1 Genes The scheme of the genes analyzed and the PCR fragments amplified by PCR are shown. Experiments were performed with the WR14-T2T (rna14-1) and BRRP-T2T (rrp6Δ) mutants and their respective isogenic wts WW-T2T and BW-T2T, all of which carried the Tho2-TAP construct. One representative polyacrylamide-gel electrophoresis is shown for each genotype. Other details are as in Figure 6. Molecular Cell 2005 18, 711-722DOI: (10.1016/j.molcel.2005.05.001) Copyright © 2005 Elsevier Inc. Terms and Conditions