(A) Bre5 and Ubp3 plus Bre5 bind RNA in vitro. Recombinant Bre5 and Ubp3 were expressed in E.coli individually and in combination, and bound to the biotinylated oligonucleotides indicated, which were immobilized on streptavidin agarose columns. (B) Tagged Bre5 and Nab3 were purified from yeast, along with a mock-purified, untagged control, and assayed for binding to labeled RNA oligonucleotides. 1: polyA (AAAAAAAAAAAAAAAAAAAAAAAAA),. 2: polyG (GGGGGGGGGGAGGGGGGGGGGAGGGGG). 3: polyU (UUUUUUUUUUUUUUUUUUUUUUUUU),. 4: polyC (CCCCCCCCCCACCCCCCCCCCACCCCC),. 5: polyA/U (AUAUAUAUAUAUAUAUAUAUAUAU),. 6: Nab3-A (AAAAAUCUUAAAUCUUAAAUCUUAAAAA),. 7 Nab3-U (UUUUUUCUUUUUUCUUUUUUCUUUUUUU),. 8: Bre5-A (AAAAAUUUGAAAUUUGAAAUUUGAAAAA),. 9: Bre5-U (UUUUUUUUGUUUUUUGUUUUUUGUUUUU). The y axis represents the signal quantified from the bound oligos (arbitrary units), the error bars are the standard deviation from three biological replicates. See also Figure 1—figure supplement 1A–C. (C) Distribution of RNA sequences recovered with Bre5-HTP and the untagged control across different RNA classes. 15-fold fewer reads were recovered with the control (Supplementary file 1, Table S2) and numbers of mapped reads are indicated below the bar graphs. (D) Metagene analysis of the distribution of RNA sequences recovered with Bre5 across protein coding transcripts (5171 features) (Xu et al., 2009), in reads per million (RPM). Sequences were aligned relative to the transcription start site (TSS). The average of 2 Bre5 CRAC replicates is shown. The standard deviation appears as a grey shadow. (E) As D, but sequences were aligned relative to the poly(A) site (pA). (F) Metagene analysis of the distribution of RNA sequences recovered with Bre5 across intron containing, protein coding transcripts (288 features), in RPM. Sequences were aligned relative to the 3’ splice site (3’SS). The average of 2 Bre5 CRAC replicates is shown. The standard deviation appears as a grey shadow. (G) As F, but data were filtered to show only genes with long exon2 regions (above 600 nt, 145 features). (H) As F, but data were filtered to show only genes with short exon2 regions (below 600 nt, 143 features).