Supplemental Methods

Strains, Growth Conditions, and Northern analysis

Strains were prepared by standard yeast genetics techniques (Gietz and Schiestl 2007). RRP6 was deleted in the BMA64 WT background using the hphMX4 marker. Site specific mutations at Reb1p binding sites and deletions of tRNA genes were made using the delittoperfettomethod (Stuckey and Storici 2013). Deletions of indicated open reading frames were obtained from the yeast knockout collection. The parent strain for anchor away (HHY168,(Haruki et al. 2008)) was modified by replacing the natMX6 marker at the FPR1 locus with the hphMX4 marker. All anchor away strains were constructed by tagging the gene of interest with the C-terminal FRB module.Unless otherwise indicated, cultures were grown in YPD (1% yeast extract, 2% bacto-peptone, 2% dextrose) at 30°C at 200 rpm and harvested at OD 0.4 to 0.6. For anchor away experiments, FRB-tagged strains were treated for 60 minutes with a 1000-fold dilution of either vehicle control (90% ethanol, 10% Tween-20) or 1 mg/mL rapamycin (final concentration of 1 μg/mL).This time point was chosen based on previous studies: Nrd1 anchor away experiments demonstrated >90% nuclear depletion of Nrd1p-FRB-GFP protein by 60 minutes while45 minutes of Rapamycin treatmentshowed more than 90% nuclear depletion for FRB-GFP tagged Nrd1p, Ysh1p, and Sen1p after 45 minutes(Schulz et al. 2013; Schaughency et al. 2014). As all previous studies to-date have shown near maximal depletion at 60 minutes, we used this time point in our studies. Due to limitations of background signal of GFP with live cell imaging, it was not possible to demonstrate 100% depletion, as low levels of nuclear protein may be sufficient to carry out that protein’s molecular functions. Therefore, we validated our NAB3, NRD1, SEN1, RRP6 and DIS3 FRB-tagged strains based on expected molecular phenotypes through 3´-end sequencing and analyzing known exosome and NNS-targets.All cultures were harvested by centrifugation at 4000 rpm for 2 minutes, washed in deionized water, and spun down in microcentrifuge tubes. The supernatant was removed and pellets were flash-frozen in liquid nitrogen and stored at -80°C. RNA extractions were performed by standard phenol/chloroform extraction and Northern blot analysis was conducted as described previously(Roy and Chanfreau 2014).

Direct RNA Sequencing analysis

Direct RNA sequencing was performed with DNase I-treated total RNA from WT and rrp6cells using the Helicos single-molecule sequencing system (SeqLL, LLC). Reads were mapped with the Heliosphere mapping software by SeqLL, LLC as previously described, allowing only uniquely mapped reads (Ozsolak and Milos 2011; Ozsolak et al. 2009; Ozsolak 2014; Geisberg et al. 2014). The 5´ ends of reads correspond to one nucleotide upstream of poly(A) sites, as the first nt upstream of the poly(A) tail is used in a “locking” step and is not sequenced (Ozsolak and Milos 2011). To account for this, 5´ ends of reads were first shifted one nucleotide downstream, and the read counts at each chromosomal coordinate were aggregated to generate bedGraphs.

3´-end poly(A)+ RNA-seq (QuantSeq) library preparation and data analysis

3´-end poly(A)+libraries were prepared with the QuantSeq 3´-end RNA sequencing kit (Lexogen, GmbH)(Moll et al. 2014). For the WT and rrp6samples, libraries were prepared per the manufacturer’s protocol. For all other samples, the reverse transcription (RT) was performed at 42°C to minimize imperfect priming between the oligo-dT primer and internal A-rich sequences. After denaturation and annealing at 42°C, a pre-warmed RT enzyme solution was added. For samples with RT performed at 42°C, 15 PCR cycles instead of 12 were performed to compensate for decreased RT yields. 50 or 100-bp sequencing was performed on the IlluminaHiSeq 2000 with the custom sequencing primer (CSP), which includes a 3´-oligo-dT stretch so that the first sequenced nucleotide corresponds to the first nucleotide upstream the poly(A) site (Moll et al. 2014). Reads were trimmed with TrimGalore v0.4.0 requiring a minimum trimmed length of 15 nucleotides. Trimmed reads were mapped using STAR v2.4.0k to the R64-2-1 version of the S.cerevisiaegenome obtained from the Saccharomyces genome database (SGD) using default mapping parameters and allowing only uniquely mapped reads (Dobin et al. 2012).5´ ends of reads at each chromosomal coordinate were aggregated to generate bedGraphs with the BEDtools v2.25.0 genomecov function(Quinlan and Hall 2010). Genome browser snapshots were derived from scalable vector graphics (.svg) files generated with the Integrative Genomics Viewer (IGV) version 2.3.52(Thorvaldsdóttir et al. 2013).

In vitropolyadenylation

10 ug of total RNA was treated with ATP and E.colipoly(A) polymerase in a total volume of 20 ul at 37°C for 30 minutes, according to manufacturer’s recommendations (NEB #M0276L). After phenol/chloroform extraction and ethanol precipitation, 5 ug of RNA were subjected to rRNA depletion with Ribo-Zero Gold rRNA Removal Kit (Illumina, Inc.), prior to 3´-end poly(A)+ library preparation.

Quality filtering of called poly(A) sites

Mapped positions were first filtered for A/G richness in the immediate genomic region downstream in accordance with previous studies on 3´-end poly(A)+sequencing (Graber et al. 2013; Wilkening et al. 2013; Gupta et al. 2014). First, poly(A) sites with six genomically encoded A nucleotides (nt) downstream (with up to two G nt) were flagged as potential internal oligo-dTmis-priming events and excluded from the analysis. Analysis of the most abundant internal priming events by-passing this filter revealed that A-richness in across an 18 bp region explained internal priming events not flagged by the 6 bp filter. Therefore, as an additional stringency filter, we excluded poly(A)-sites with 12 or more adenosines present in these downstream 18 nt. On the order of ~1% of sequencing reads involving long stretches of T’s, possibly due to priming internal to the poly(A) tail or due to a low level of contamination of the sequencer with Illumina Read 1 primer. As an additional quality control step, poly(A) sites were flagged if the preceding 15 or more nucleotides were adenosines. The total reads for each bedGraphfile after filtering were normalized to 1 million.

Cluster annotation

Annotations were obtained from the Saccharomyces Genome Database (SGD) using the GFF annotations file for the S288C reference genome (version R64-2-1)(Cherry et al. 2012; Engel et al. 2014). 3´-UTR end coordinates were re-annotated using transcript isoform sequencing data (TIF-seq) by selecting the 3´-most coordinate with respect to the stop codon accounting for 90% of the total ORF-spanning reads(Pelechano et al. 2013). For snoRNAs, the MRP snoRNANME1 is neither box C/D nor box H/ACA and was omitted from the analysis. The snR17a/b genes, which encode the U3 box C/D snoRNA, were omitted from the box C/D analyses because they are 3´-end processed by the yeast RNase III homolog Rnt1p independently of the NNS pathway (Kufel et al. 2000). Annotations for CUTs, SUTs, and XUTs were obtained from the pyCRAC software package and remapped to R64-2-1 coordinates with the NCBI remap tool (Webb et al. 2014).

Data processing on previous datasets and calling motif sites

Raw reads from NET-seq data from WT and dst1Δwere obtained from NCBI Gene Expression Omnibus (GEO) accession number GSE25107(Churchman and Weissman 2011). Reads were mapped with the same parameters as for the poly(A) site sequencing but omitting the downstream A/G filtering step. DNase I hyper-resistant sites (DRS) were obtained from SGD(Hesselberth et al. 2009; Cherry et al. 2012). In vivo and in vitro nucleosome occupancies were obtained from SGD (Kaplan et al. 2009). To obtain experimentally verified Reb1p binding sites, DNase I hyper-resistant regions, Reb1-ChIP exo sites(Rhee and Pugh 2011), and Reb1p ORGANIC ChIP(Kasinathan et al. 2014)were examined for sequences with up to two nucleotides deviation from the consensus binding motif TTACCCG, resulting in the identification of 1270 Reb1p sites from the union of these datasets. 896 Abf1p binding sites were obtained by scanning Abf1p ORGANIC ChIP(Kasinathan et al. 2014) and DNase I hyper-resistant regions for the motif TNNCGTNNNNNNTGAT with up to two mismatches allowed. 881 Rap1p binding sites were obtained by scanning Rap1p bound regions determined by competition-ChIP, ChIP-exo and DNase I hyper-resistant regions for the closest match to the consensus Rap1p binding site ACACCCATACAT, taking the union of binding sites from both datasets (de Boer and Hughes 2012; Lickwar et al. 2012; Rhee and Pugh 2011). Potential binding sites for Mcm1p (CCNNWTYRGGAA), Tbf1p (TTAGGG), and Cbf1p (CACGTG) were obtained from by scanning the entire genome for sites with up to 1 mismatch from these consensus motifs. Okazaki fragment raw reads were obtained from GEO accession number GSE33786 and mapped to the R64-2-1 version of the S.cerevisiaegenome. The 3´-ends of Okazaki fragments correspond to the 5´-ends of read 2 for the paired end reads in this study, as IlluminaTruseq primer 2 adapters were ligated to DNA 3´-OH ends (Smith and Whitehouse 2012). The 3´-ends were processed in the same pipeline described for RNA 3´-ends in this study.

Supplemental References

Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, Christie KR, Costanzo MC, Dwight SS, Engel SR, et al. 2012. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res40: D700–D705.

Churchman LS, Weissman JS. 2011. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature469: 368–373.

de Boer CG, Hughes TR. 2012. YeTFaSCo: a database of evaluated yeast transcription factor sequence specificities. Nucleic Acids Res40: D169–179.

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. 2012. STAR: ultrafast universal RNA-seq aligner. Bioinformatics bts635.

Engel SR, Dietrich FS, Fisk DG, Binkley G, Balakrishnan R, Costanzo MC, Dwight SS, Hitz BC, Karra K, Nash RS, et al. 2014. The reference genome sequence of Saccharomyces cerevisiae: then and now. G3 Bethesda Md4: 389–398.

Geisberg JV, Moqtaderi Z, Fan X, Ozsolak F, Struhl K. 2014. Global Analysis of mRNA Isoform Half-Lives Reveals Stabilizing and Destabilizing Elements in Yeast. Cell156: 812–824.

Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc2: 31–34.

Graber JH, Nazeer FI, Yeh P, Kuehner JN, Borikar S, Hoskinson D, Moore CL. 2013. DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast. Genome Res23: 1690–1703.

Gupta I, Clauder-Münster S, Klaus B, Järvelin AI, Aiyar RS, Benes V, Wilkening S, Huber W, Pelechano V, Steinmetz LM. 2014. Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions. Mol Syst Biol10: 719.

Haruki H, Nishikawa J, Laemmli UK. 2008. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol Cell31: 925–932.

Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, et al. 2009. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods6: 283–289.

Kaplan N, Moore IK, Fondufe-Mittendorf Y, Gossett AJ, Tillo D, Field Y, LeProust EM, Hughes TR, Lieb JD, Widom J, et al. 2009. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature458: 362–366.

Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S. 2014. High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods11: 203–209.

Kufel J, Allmang C, Chanfreau G, Petfalski E, Lafontaine DL, Tollervey D. 2000. Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La binding. Mol Cell Biol20: 5415–5424.

Lickwar CR, Mueller F, Hanlon SE, McNally JG, Lieb JD. 2012. Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function. Nature484: 251–255.

Moll P, Ante M, Seitz A, Reda T. 2014. QuantSeq 3′ mRNA sequencing for RNA quantification. Nat Methods11. (Accessed August 9, 2016).

Ozsolak F. 2014. Quantitative polyadenylation site mapping with single-molecule direct RNA sequencing. Methods Mol Biol Clifton NJ1125: 145–155.

Ozsolak F, Milos PM. 2011. Transcriptome profiling using single-molecule direct RNA sequencing. Methods Mol Biol Clifton NJ733: 51–61.

Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, Thompson JF, Bowers J, Jarosz M, Milos PM. 2009. Direct RNA sequencing. Nature461: 814–818.

Pelechano V, Wei W, Steinmetz LM. 2013. Extensive transcriptional heterogeneity revealed by isoform profiling. Nature497: 127–131.

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics26: 841–842.

Rhee HS, Pugh BF. 2011. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell147: 1408–1419.

Roy K, Chanfreau G. 2014. Stress-induced nuclear RNA degradation pathways regulate yeast bromodomain factor 2 to promote cell survival. PLoS Genet10: e1004661.

Schulz D, Schwalb B, Kiesel A, Baejen C, Torkler P, Gagneur J, Soeding J, Cramer P. 2013. Transcriptome surveillance by selective termination of noncoding RNA synthesis.Cell155: 1075–1087.

Smith DJ, Whitehouse I. 2012. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature483: 434–438.

Stuckey S, Storici F. 2013. Gene knockouts, in vivo site-directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae. Methods Enzymol533: 103–131.

Thorvaldsdóttir H, Robinson JT, Mesirov JP. 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform14: 178–192.

Webb S, Hector RD, Kudla G, Granneman S. 2014. PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast. Genome Biol15: R8.

Wilkening S, Pelechano V, Järvelin AI, Tekkedil MM, Anders S, Benes V, Steinmetz LM. 2013. An efficient method for genome-wide polyadenylation site mapping and RNA quantification. Nucleic Acids Res41: e65.