Yra1 influences the cleavage-polyadenylation apparatus. (a) Clp1 disrupts the Pcf11-Yra1 interaction. GST-Yra1 beads (full length F.L.) bound to Pcf11(ZCZ-HisHis6) were incubated with RNAse-treated Clp1 (0.25–16µg) or buffer (lane 3), and Pcf11(ZCZ-His₆) released into the supernatant was immunoblotted (top panel). Coomassie stained GST-Yra1 is a loading control (bottom panel). (b) Sub2 and RNA disrupt the Pcf11-Yra1 interaction. Immunoblot of released Pcf11(ZCZ-His₆) as in panel a. (c) Yra1 inhibits coupled cleavage-polyadenylation of ³²P-labeled GAL7 RNA precursors (lane 4) (see Methods). GST-Yra1F.L. or GST-Yra1RRM (5 µg) was added in lanes 2, 3. Products were quantified by Phosphorimager, and ratios of polyadenylated products:precursor calculated. Values are means, ± s.e.m. (d) Yra1 inhibits uncoupled cleavage and polyadenylation. Left: processing of GAL7 + cordycepin to inhibit polyadenylation. Right: polyadenylation of pre-cleaved GAL7 + ATP.

Distinct Chip profiles for export and 3’ processing factors. (a) Yra1 occupancy decreases as Clp1 occupancy increases at the 3’ ends of genes. Average distributions of Pcf11, Clp1, Yra1 and Sub2 normalized to the values at the 3’ end of the ORF on 789 protein-coding genes (Supplementary Table 1). Plot includes the ORF divided into 10 equal intervals and 1 kb of 5’ and 3’ flank. Variation between these proteins in relative signals upstream of the ORF after normalization results from differences in average signal:background ratios between the IPs. Note distinct transitions of export (solid arrow) and 3’ processing factors (dashed arrow) at 3’ ends. Log2 ChIP signals relative to input DNA are shown for ORFs (dotted lines, red arrow) with 1 kb of 5’ and 3’ flanking sequence (smooth lines). (b) Representative ChIP profiles of Pcf11, Clp1, and Yra1 generated with ChIPViewer⁴³. Each point is the average ChIP signal at 20-base intervals. Note distinct profiles of Pcf11 and Clp1 recruitment. (c) Average distributions as in (a) showing enrichment of Yra1, Sub2 and Clp1 on non-coding RNA genes as described ⁴³.

Yra1 depletion enhances Clp1 recruitment on long genes. (a) Depletion of Yra1 in glucose grown GAL1-Yra1 cells (DBY1276-2). Immunoblots of Yra1 and Pgk1 loading control are shown. (b) Average ChIP-Chip profiles as in Fig. 2a of Pcf11 and Clp1 normalized to the values at the 5’ end of the ORF on 128 long genes (>2000 bases, Supplementary Table 2) in isogenic WT and Yra1-depleted cells (8 hr in glucose). (c) Representative ChIP-Chip profiles of Pcf11 and Clp1 in WT (top row) and Yra1-depleted (bottom row) cells. Note enhanced Clp1 occupancy relative to Pcf11 within the ORFs in Yra1-depleted cells.

Yra1 depletion affects alternative poly(A) site choice at ACT1. (a) RNAse protection assay of total RNA from WT and Yra1-depleted GAL1-YRA1 cells (8 hr glucose as in Fig. 3a). A map of the major poly(A) sites is shown below. (b) Reduced ORF distal and enhanced proximal site usage in Yra1-depleted cells. Cleavage at each site is expressed as a fraction of total signal after normalization for the ³²P-U content of protection products 1–5. Values are means, ± s.e.m. (c) 3’ RNA-seq (see Methods) confirms the shift in favor of ORF proximal ACT1 poly(A) sites in isogenic WT and Yra1-depleted (Yra1dep) cells (8 hr in glucose, see Fig. 3a) and in the Helicos data set ⁴¹. (−) refers to transcripts on the Crick strand. The Helicos track combines reads on both strands.

Widespread effects of Yra1 depletion on alternative poly(A) site choice within 3’ UTRs. (a, b) Genome browser views of RNA-seq reads across poly(A) sites in WT and Yra1-depleted GAL1-YRA1 cells (8 hr in glucose) and in the Helicos data set ⁴¹. (+) and (−) refer to transcripts of the Watson and Crick strands respectively. ORF proximal and ORF distal shifting of poly(A) site choice in Yra1-depleted conditions are shown in (a) and (b) respectively. Total read counts are 2.70 million for WT and 1.58 million for yra1-dep data sets.

Yra1 depletion alters use of poly(A) sites within genes and at ncRNAs. (a–e) Genome browser views of RNA-seq reads across poly(A) sites in WT and Yra1-depleted cells as in Fig. 5. (a, b) Premature poly(A) sites within ORFs that are reduced (a) or enhanced (b) by Yra1 depletion. (c–e) Altered poly(A) site use at ncRNAs following Yra1 depletion at SUTs/CUTs (c), snoRNAs (d) and regulatory ncRNAs (e).

Positioning of consensus efficiency element differs between “Yra1 sensitive” and “insensitive” poly(A) sites. (a) Distribution of positioning and efficiency element (PE and EE) consensus sites relative to cleavage sites (black arrowhead, position 0) used in WT cells on genes that are unaffected by Yra1 depletion (non-shifters, 2782 genes). When multiple elements were present, we chose the closest to the cleavage site. Map shows location of poly(A) site consensus elements including U-rich sequences (Ur). Red and green arrowheads mark poly(A) sites shifted upon Yra1-depletion. (b) Distributions of EE motifs relative to cleavage sites used in WT cells at genes whose major poly(A) sites shift ≥15 bases following Yra1 depletion. Note the divergent positions of EE elements at genes that change their poly(A) site choice ORF proximally (349 genes, red line) or distally (381 genes, green line). (c) Distributions of EE motifs relative to cleavage sites used in Yra1-depleted cells at the same genes shown in (b). Note the change from WT cells (b) in the position of EE elements (arrows) relative to cleavage sites. (d) A regulated assembly model for how the export factor Yra1 could influence co-transcriptional 3’ end processing. Left and right panels depict recruitment of Yra1 and CF1A subunits within genes and at 3’ ends. Rna14 and Rna15 are dotted at left to indicate that the timing of their recruitment relative to Pcf11 is not known. 'Poly(A) site cleavage (scissors, right panel) becomes possible at 3’ ends only after assembly of the complete CF1A complex.