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. 2011 May 18;30(10):1953-64.
doi: 10.1038/emboj.2011.109. Epub 2011 Apr 8.

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors

Cristina Tous  1 , Ana G Rondón, María García-Rubio, Cristina González-Aguilera, Rosa Luna, Andrés Aguilera
Affiliations

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors

Cristina Tous et al. EMBO J. .

Abstract

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of the G-less cassette-based run-on transcription-elongation assay (GLRO) in wild-type and spt4Δ and rpb9Δ mutants. (A) Scheme of the tandem G-less cassette constructs used for GLRO analysis. Black rectangles represent sequences derived from CYC 3′-flanking region, and the white and grey rectangles correspond to the two G-less cassettes and the 2-kb lacZ fragment, respectively. (B) GLRO analysis of wild-type and spt4Δ and rpbΔ9 mutants transformed with the GLRO-short and GLRO-long systems. Transformants were grown in SC-leu medium to exponential phase and run-on transcription assays were performed as described in Materials and methods. The transcription run-on products were digested with RNase T1 and resolved in a 6% PAGE. A representative acrylamide gel is shown. For each sample, the ratio of total counts incorporated into the distal versus the proximal G-less cassette was normalized against the ratio for the same construct in the wild-type strain. The mean value and s.d. of three independent experiments are shown.
Figure 2
Figure 2
GLRO analysis of PAF mutants. Wild-type (BY4741), paf1Δ, rtf1Δ, cdc73Δ and leo1Δ isogenic strains were transformed with the GLRO-short and GLRO-long constructs and transcription run-on assays were performed. A representative assay is shown. The mean value and s.d. of three independent experiments are shown. Other details are as in Figure 1.
Figure 3
Figure 3
Analysis of transcription elongation in THSC. (A) GLRO analysis of THSC mutants. Transcription run-on assays of thp1Δ, sac3Δ, sus1Δ, mutants carrying the plasmids GLRO-short and GLRO-long. (B) RNAPII occupancy in sus1Δ mutant. ChIP analyses in wild-type (BY4741), thp1Δ and sus1Δ strains transformed with the pLAUR expression system. ChIP analyses in wild-type, thp1Δ and sus1Δ strains carrying the GAL1p::YLR454w fusion construct located at the endogenous YLR454w chromosomal locus. The scheme of the gene and the PCR-amplified fragments are shown. Numbers indicate the primer position respect to the first ATG of the gene. The sequence is provided in Supplementary Table II. The DNA ratios between the 5′ and 3′ regions were calculated from their signal relative to the signal of the intergenic region. The recruitment data shown refer to the value of the 5′ region normalized to 100%. ChIPs were performed from three independent cultures, and quantitative PCRs were repeated three times for each culture.
Figure 4
Figure 4
GLRO analysis of mutants of the SAGA, nuclear exosome and 3′-end processing factors. (A) GLRO analysis of SAGA mutants. Transcription run-on assays of wild-type, sgf73Δ, gcn5Δ and spt20Δ strains carrying GLRO-short and GLRO-long plasmids are shown. (B) GLRO analysis of nuclear exosome and 3′-end processing mutants. rrp6Δ, rna14-1, rna15-1, and a isogenic wild-type strain were transformed with the GLRO-short and GLRO-long plasmids and transcription run-on assays were performed. A representative assay is shown. The mean value and s.d. of three independent experiments are shown. Other details are as in Figure 1.
Figure 5
Figure 5
Genetic interactions of Nup84 with transcription-elongation and mRNA export factors. (A) Tetrad analyses of different crosses between nup84Δ and dst1Δ, spt4Δ and sac3Δ strains. Circles indicate the positions of the double mutants. Only the double nup84Δ sac3Δ are inviable. (B) Micophenolic acid (MPA) sensitivity of nup84Δ, dst1Δ and spt4Δ single and double mutants as determined by 10-fold serial dilutions on SC plates containing the indicated concentrations of MPA.
Figure 6
Figure 6
RNAPII distribution along different genes in nucleoporin mutants. (A) RNAPII ChIP analysis in the pLAUR system in the nup84Δ and an isogenic wild-type strains. (B) RNAPII ChIP analysis in the HSP104 endogenous gene in the nup84Δ, thp1Δ and spt4Δ mutants and their isogenic wild-type strain (C) RNAPII ChIP analysis in the GLRO-long system in the nup84Δ, thp1Δ and spt4Δ mutants and their isogenic wild-type strain. Other details are as in Figure 3.
Figure 7
Figure 7
GLRO analysis of nucleoporin mutants. (A) GLRO analysis was performed in nup84Δ, nup133Δ, nup60Δ and an their isogenic wild-type strain on both the GLRO-short and GLRO-long systems. The mean value and s.d. of three independent experiments are shown. Other details are as in Figure 1. (B) In vitro transcription-elongation assays of wild-type and nup84Δ WCEs. Scheme of the two G-less cassette systems pGCYC1-402 used for the in vitro transcription-elongation assay. RNase T1 treatment of the mRNA driven from the GAL4-CYC1 promoter renders two fragments corresponding to the two G-less cassettes. In vitro transcription assays of WCEs from BY4742, nup84Δ and thp1Δ isogenic strains are shown. Each reaction was stopped after 40 min, treated with RNaseT1, and run in a 6% PAGE. Efficiency of transcription elongation was determined as the percentage of total transcripts that covered the 376-nt G-less cassette with respect to the transcripts that covered the 84-nt cassette. The signal was normalized with respect to the U content of each G-less cassette (161 U residues in the long cassette and 24 in the short one). The mean value and s.d. of at least three independent experiments are shown for WT and nup84Δ.
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