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. 2017 Oct;23(10):1512–1524. doi: 10.1261/rna.061986.117

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© 2017 Hálová et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 4. — prp45(1–169) delays cotranscriptional spliceosome assembly. ChIP-qPCR analysis of cotranscriptional recruitment of spliceosomal components was performed on the ECM33 and ACT1 genes (A,J; positions of PCR amplicons are indicated). Cells expressing HA-tagged Prp45 or Prp45(1–169) were used for ChIP-qPCR with the anti-HA antibody. Signals were normalized to input values and expressed as the fold enrichment relative to the signal from exon 1 in the Prp45-HA strain. The strain without the tagged protein served as a negative control (B,K). RNA Pol II ChIP signals were obtained using the anti-Rpb3 antibody. Cotranscriptional spliceosome assembly was monitored via the recruitment of HA-tagged components of commitment complex 1 using HA-tagged Prp42 (D,M), Msl5 (E,N), and Mud2 (F,O). Subsequent stages of spliceosome formation were followed using HA-tagged members of U2 snRNP (Msl1; G,P), U5 snRNP (Brr2; H,Q), and NTC (Prp19; I,R). Signals were normalized to input values and expressed as the fold enrichment relative to the signal obtained from the telomeric region (TELVIR; graphs C–I and L–R). Error bars represent SD from at least four independent experiments.