Fig. 1: Xrn2-Rai1 complex interacts with Spt5-Spt4 containing Pol II transcription complexes.
a Volcano plot highlighting proteins enriched in Spt5-Flag purifications. The top of the figure depicts the Spt5 domain organisation. Numbers correspond to the positions of amino acids. Statistical analysis was performed using empirical Bayes-moderated t-statistics (n = 2). b Xrn2 associates with Spt5 complexes in a phosphorylation-independent manner. Proteins enriched in purifications of Spt5 from WT Spt5, T1A, T1E and cells lacking PP1 (Dis2) phosphatase are presented as dot plots, and circle size reflects a number of peptides and colour corresponds to enrichment over mock IP. Proteins are grouped according to enrichment (panel 1 – present in all, 2 – decreased in T1E or dis2Δ, 3 – reduced in T1A or WT, 4 – more abundant in T1A, 5 – unassigned). Metabolic enzymes and ribosomal proteins were filtered out. c Xrn2-Rai1-Spt4/5-Pol II-DNA/RNA form a stable complex. Scaffold 1 used is listed in Supplementary Data 2 (compare Supplementary Fig. 2a) as previously published35. The reconstituted complex was subjected to size exclusion chromatography (Superose 6) and analysed by SDS-PAGE. mAU represents milli-absorbance units (n = 2). d Xrn2 alignment shows conserved catalytic residues that have been mutated in Xrn2M (D237A for in vitro or D235A for in vivo experiments). e In vitro Xrn2 RNA degradation assay. 3’ FAM-labelled 5’-monophosphate-RNA (P-RNA-FAM, 26 nt) substrate was incubated with either Xrn2, Xrn2M (D237A) or buffer for the indicated time. Intact RNA substrate and degradation products (indicated on the side) were resolved on 10% 8 M UREA-PAGE gel (n = 2).
