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. 2013 Aug 15;154(4):775-88.
doi: 10.1016/j.cell.2013.07.033. Epub 2013 Aug 8.

From Structure to Systems: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II

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From Structure to Systems: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II

Hannes Braberg et al. Cell. .
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Abstract

RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ∼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

Figures

Figure 1
Figure 1. Generation and Selection of RNAPII Point Mutants
(A) RNAPII point mutants were screened for three transcription-related phenotypes. (GalR, left) Deletion of the major GAL10 p(A) site (gal10Δ56) results in RNAPII readthrough and interference with initiation at GAL7, causing a Gal-sensitive phenotype. GalR mutants increase GAL10 3′ end formation/termination, thereby rescuing GAL7 expression. (MPA, middle) Mycophenolic acid (MPA) inhibits IMP-dehydrogenase (IMPDH)-dependent GTP synthesis but is counteracted by upregulation of an MPA-resistant form of IMPDH, IMD2. Transcriptional defects that are sensitive to low GTP levels or reduce IMD2 expression render cells sensitive to MPA. (Spt, right) Insertion of a Ty retrotransposon into LYS2 (lys2-128∂) results in a lysine auxotrophy due to transcription block. Certain mutants suppress lys2-128∂ and allow expression of LYS2 due to activation of an internal promoter. Spot tests to identify each phenotype for three representative mutants are displayed. (B) Positions, mutations, and phenotypes of the 53 single point mutants analyzed in the pE-MAP. Colored lines represent subunit sequences, with mutations denoted by residue numbers and single letter amino acid codes for WT and mutant. See also Figure S1 and Table S1.
Figure 2
Figure 2. pE-MAP Interactions Span Numerous Biological Processes, Depend on Spatial Location of Mutated Residues, and Are Not Direct Consequences of Changes in Gene Expression
(A) ROC curves comparing the power of genetic profile correlations from the pE-MAP (red) and an E-MAP focused on chromosome biology (Collins et al., 2007b) (blue) to predict physical interactions between pairs of proteins (Collins et al., 2007a; Experimental Procedures). AROC, area under the curve. (B) Genetic profile correlations between pairs of mutated residues compared to the three-dimensional distance between their α carbons. Blue points denote residue pairs within the same RNAPII subunit; red points represent pairs in different subunits. Negatively correlated residue pairs were excluded, as were four mutants of residues absent from the coordinate file (PDB ID: 2E2H) (Rpb1 D1502, Rpb7 V101, Rpb7 D166, and Rpb7 L168) (Wang et al., 2006). Correlations between 0 and 0.2 are dimmed to highlight trends at higher correlations. (C) Effect of RNAPII point mutations on gene expression, compared to the corresponding genetic interaction scores between RNAPII mutants and deletion/DAmP alleles. All combinations of the 26 RNAPII mutants and 1,192 library genes/mutants that were examined via both pE-MAP and expression analyses are included. No global changes in gene expression were observed (measured by spike-in control RNA; Experimental Procedures). (D) Comparison between pairwise RNAPII mutant correlations of genetic interaction profiles and gene expression profiles. See also Figure S2, Table S1, Table S2, and Data S1.
Figure 3
Figure 3. Comparison of the pE-MAP with Previously Collected Genetic Interaction Data Reveals Functional Associations between RNAPII Residues and Protein Complexes
(A) Module map depicting genetic similarity of RNAPII mutants with genes encoding the indicated protein complex subunits (Experimental Procedures). Edge widths correspond to statistical significance of connections. Only RPB1 edges with a false discovery rate <0.1 are displayed. Four mutated residues linked to the kinetochore are highlighted in blue, and the blow-up indicates their structural locations. (B) Nineteen mutants were examined using a chromosome transmission fidelity (CTF) assay. The four kinetochore-linked mutants highlighted in (A) exhibit chromosome loss in >15% of their colonies (blue bars), whereas unlinked mutants display no or weak phenotype (red bars, representative set). See also Figure S3 and Table S3.
Figure 4
Figure 4. pE-MAP Profiles Differentiate between Subtle Changes in Transcription-Related Phenotypes and Identify RNAPII Mutations that Affect Start Site Selection
(A) pE-MAP clustering in relation to MPA and Spt phenotypes of alleles. The RNAPII alleles are clustered by pE-MAP profiles, and their colors indicate degrees of MPA and Spt phenotypes (determined from the spot tests). (B) Effect of RNAPII mutations on start site selection at ADH1 determined by primer extension analysis. The heatmap describes the fractional change of start site in each bin of the ADH1 schematic (bottom). (C) Rpb1 I1327 and Rpb1 S713 connect to the TL (magenta). Mutations in I1327 could affect the structural region of the TL (E1103) via a network of loops and helices in Rpb1 (gray), and S713 is close to the TL catalytic site in its open conformation, via a Rpb9 loop (orange). In particular, the proline substitution, S713P, could result in structural changes affecting the TL. Coordinates for TL, Rpb9, and the S713 loop are from PDB ID 1Y1V (Kettenberger et al., 2004), and all others are from 2E2H (Wang et al., 2006). The bridge helix is shown in cyan; template DNA, blue; nontemplate DNA, green; and RNA, red. The incoming GTP base is colored by atom. See also Figure S4, Table S4, and Data S1.
Figure 5
Figure 5. pE-MAP and Expression Profiles Are Indicative of Biochemical Activity
(A) In vitro transcription rates from (Kaplan et al., 2012) and in vivo growth rates relative to WT for RNAPII active-site mutants. The dendrogram was generated via hierarchical clustering of the genetic profiles. Error bars represent 95% confidence intervals for transcription rates and SD for growth rates. Means and SD of growth rates were derived from three technical replicates. Note that rpb1 G1097D was too sick for reproducible E-MAP analysis. (B) In vitro transcription rate difference between pairs of active-site mutants in relation to their genetic and expression profile correlations. (C) Residues H1085 and F1086 reside in the catalytic site of the TL, whereas E1103 is part of the distal flanking α helix that structurally constrains the TL in open conformations. TL is shown in green (closed) and magenta (open); template DNA, blue; and RNA, red. The incoming GTP base is colored by atom. Coordinates for open TL are from PDB ID 1Y1V, and all others are from 2E2H. (D) Counts of high-scoring interactions (pE-MAP score >3.3 [97.5 percentile] or <−5.1 [2.5 percentile]) in the complete genetic profiles or changes >1.7-fold in the genome-wide expression profiles of the indicated RNAPII mutants. In vitro transcription rates are indicated on the scale on the right. See also Figure S5.
Figure 6
Figure 6. Effects of Altering RNAPII Transcription Rate on In Vivo Splicing Efficiency
(A) (Top) Microarray schematic for each intron-containing gene: probe I (intron) hybridizes to premRNA, J (junction) to mature mRNA, and E (exon) to both. (Center) Heatmap of I/J log2 ratios for the slow (rpb1 H1085Q and F1086S) and fast (rpb1 E1103G and G1097D) mutants, corresponding to the enrichment of pre-mRNA over mature mRNA. The side panels highlight a subset of genes that behave reciprocally in fast and slow RNAPII backgrounds. (B) Number of genes exhibiting >20% change in pre-mRNA-to-mature mRNA ratio (bars, scale on left) and median I/J log2 ratio (asterisks, scale on right) across entire array. MPA treatment was 10 min; WT denotes competitive hybridization between two WT cultures. I/J denotes I/J log2 ratio. See also Figure S6 and Table S5.
Figure 7
Figure 7. Genetic Interaction Patterns with Fast and Slow RNAPII Mutants Reveal Sub1 as a Transcription Factor that Regulates Start Site Selection and Influences mRNA Splicing
(A) Genetic profiles of library mutants, sorted on the difference between their average interaction with fast and slow RNAPII mutants (Table S6). (B) Patch tests examining the sensitivity of slow TL mutants to sub1Δ. WT RPB1 plasmid covering rpb1 mutants in left panel is lost in right panel. (C) Spot tests examining the effect of sub1Δ on fast mutants in absence (left) and presence (right) of MPA. (D) Comparison of sub1Δ effect on gene expression in rpb1 E1103G (difference between red and blue) and WT (difference between green and y = 0). Included are all array transcripts exhibiting a >1.5-fold expression change in at least one of the three mutants. Transcripts are sorted by expression change in E1103G. (E) Primer extension at ADH1 to map transcription start sites for rpb1 F1086S, E1103G, and sub1Δ (Figure S7C). Bar colors correspond to sequence windows in the ADH1 schematic (top), and heights specify the mean fraction change of transcription start in mutant compared to WT. Error bars represent SD. (F) Splicing microarray analysis of sub1Δ, as in Figure 6 (Table S5). Number of genes exhibiting >20% change in pre-mRNA-to-mature mRNA ratio (bars, scale on left) and median I/J log2 ratio (asterisks, scale on right). (G) Model for the effect of Sub1 and RNAPII activity on start site selection and splicing. Fast RNAPII mutations (class I) result in upstream transcription start and diminished splicing efficiency, whereas sub1Δ or slow RNAPII mutations (class II) shift transcription start downstream and enhance splicing. See also Figures S6 and S7 and Tables S4, S5, and S6.

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