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Molecular Cell, Volume 45

Supplemental Information

Decapping of Long Noncoding RNAs

Regulates Inducible Genes Sarah Geisler, Lisa Lojek, Ahmad M. Khalil, Kristian E. Baker, and Jeff Coller Supplemental Experimental Procedures Yeast Strains and Growth Conditions All yeast strains used in this study are in the BY4741 genetic background unless otherwise noted, the genotypes of all strains used are listed in Table S4 below. Strains constructed in this study were prepared by standard methods (Brachmann et al., 1998; Longtine et al., 1998). Unless otherwise indicated, cells were grown at 24 oC into mid-log phase (3.0 X 107 cells ml-1) in standard synthetic complete medium (pH 6.5) with the appropriate amino acids supplemented and either 2% glucose, 2% raffinose, or 2% galactose. Cultures used for RNA-seq were grown in glucose media. Cells for the MFA2 reporter transcriptional shut-off assays were grown in 2% galactose 1% sucrose into mid-log phase then transferred to no sugar media and glucose was added to 4%. For galactose induction/ GAL10 lncRNA shut-off assays cells were grown to mid-log phase in 2% raffinose then transferred to no sugar media and galactose was added to a final concentration of 4%. For TSA treated inductions, cells were grown as above except that TSA was added to the growth media for all cultures at a final concentration of 10μM. The GAL10 lncRNAΔ and dcp2Δ/GAL10 lncRNAΔ strains (yJC960 and yJC962) were constructed by deleting GAL10 by standard methods and then transforming in GAL10 plasmids with the Reb1p binding site mutations described by Houseley et al., 2008. The GAL4 lncRNAmut strains (yJC824 and yJC1281) were made by introducing a C-terminal HA tag on the genomic copy of GAL4 in WT (yJC151) or dcp2Δ (yJC327) strains respectively (Longtine et al., 1998). Introduction of the tag disrupted elements necessary for full expression. Strains yJC424 and yJC425 were constructed by transforming plasmids expressing dcp2-4-HA and DCP2-HA respectively into yJC327. Plasmids and Oligonucleotides All plasmids and oligonucleotides used in this study are listed in Table S5 and S6 respectively. pJC317 was constructed by PCR amplifying 500bp up and downstream of the DCP2-HA loci in a chromosomally HA tagged strain (yJC411) introducing XbaI sites. The resulting DCP2-HA fragment was cloned into pJC70 (YCplac111) a centromeric expression vector to make pJC317. pJC318 was constructed from pJC317 by site-directed mutagenesis to introduce the dcp2-4 point mutations. All oligonucleotides used to construct pJC317 and pJC318 are listed in Table S5. For pJC409 a region of GAL10 approximately 500bp up and downstream was PCR amplified with XbaI and BamHI sites flanking and cloned into pJC69 (YCplac33) a centromeric yeast expression vector. pJC422 was made from pJC409 by disrupting four REB1 binding sites within GAL10. The consensus and three near consensus REB1 binding sites found within the 3‘ end of

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GAL10 were mutated by site-directed mutagensis according to the Stratgene QuikChange Site-Directed Mutagenesis Kit. All oligonucleotides used to construct pJC409 and pJC422 are listed in Table S6. Riboprobe templates for GAL mRNAs and lncRNAs were made by dropping restriction fragments into pBluescript SK+ (see Table S5). RNA-Seq Library construction Total RNA was isolated from yeast cells using standard methods. Libraries were prepared with minimal deviation according to Illumina’s Directional mRNA-Seq Sample Prep Guide (Part # 15018460 Rev. A) using components of the mRNA-seq and small RNA Sample Preparation Kits. Briefly, from 10ug of total RNA contaminating DNA was removed by using the QIAGEN DNase Set upon clean up over a QIAGEN RNeasy column. We omitted poly(A) selection as a means to reduce ribosomal RNA reads in order not to bias our analysis toward poly(A) RNA. RNA was fragmented according to Illumina’s recommendations (5 minutes at 94 oC in 1X fragmentation buffer). The fragmented RNA was purified over an RNeasy MinElute spin column using a modified procedure such that appropriately sized RNA was purified. The ends of the RNA were repaired with phosphatase treatment (NEB) followed by PNK treatment (NEB) as per Illumina’s instructions. The fragmented and end repaired RNA was then purified over another RNeasy MinElute spin column with modifications to the procedure to again insure that appropriately sized RNA was obtained after purification. The 3’ (v1.5 sRNA 3� adapter) and 5’ (SRA 5� Adapter) adapters supplied in the small-RNA sample preparation kit were ligated onto the RNA. The 3’ adapter was ligated with T4 RNA Ligase2, truncated (NEB) as per Illumina’s instructions and the 5’ adapter was ligated with T4 RNA Ligase (NEB). The resulting adapter ligated RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen) and the SRA RT Primer. The library was amplified using Phusion DNA Polymerase and the Illumina supplied GX1 and GX2 primers by PCR (12 cycles). The resulting library was gel purified and sent out for single end read sequencing. The library was sequenced at the Applied Genomics Technology Center at Wayne State University. RNA-seq Data Analysis FASTA files containing raw reads were uploaded to Galaxy and the majority of the downstream analysis was carried out using Galaxy (http://main.g2.bx.psu.edu; Goecks et al., 2010; Blankenberg et al., 2010; Giardine et al., 2005). Raw reads were mapped to the yeast genome with Bowtie to obtain 84.4 and 61.2 million mapped reads for WT and dcp2Δ libraries respectively. Ribosomal RNA reads were digitally filtered out using a selection tool that utilized a regular expression such that reads that were not mapped to the ribosomal DNA locus were retained. This resulted in 5.2 million and 5.5 million non-ribosomal RNA reads for WT and dcp2Δ libraries respectively. Reads mapped to the yeast genome were displayed on the UCSC Genome Browser (http://genome.ucsc.edu/; Kent et al., 2002; Fujita et al., 2010; Karolchik et al., 2004) and manually curated. To make the panels in Figure 1, reads were separated by strand in Galaxy and downloaded for further manipulation using the integrative genomics viewer (IGV) (Robinson et al., 2011). Index and count files were generated using igvtools and displayed in IGV. RNA Analysis

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For Northern and quantitative real time PCR (RT-PCR) analysis total RNA was isolated from yeast cells using standard methods. Yeast cell pellets with resuspended in LET buffer and cells were lysed by bead bashing with glass beads in the presence of phenol buffered with LET. Nucleic acids were isolated by extraction sequentially with phenol/chloroform buffered with LET then chloroform. Nucleic acids were ethanol precipitated with sodium acetate and resuspended in water. 20ug (galactose induction and GAL10 lncRNA shut-off analysis), 40ug (steady-state analysis), or 60ug (PHO84 lncRNA steady-state analysis) of total RNA was loaded per lane on 1.4% formaldehyde agarose gel and separated by electrophoresis. The RNA was transferred to nitrocellulose membranes and Northern analysis was done with radiolabelled probes. The oligonucleotides and plasmids used to make the probes are listed Tables S5 and S6. Specifically, riboprobes for GAL10, GAL1, GAL2, and GAL4 lncRNAs and GAL2 and GAL4 mRNAs probes were generated from plasmids containing restriction fragments of their respective genes. Linearized plasmids were in vitro transcribed with either T7 or T3 RNA polymerase in the presence of alpha labelled UTP 32P. The PHO84 lncRNA, GAL1 mRNA, GAL10 mRNA, and SCR1 RNA probes were single stranded DNA oligonucleotide primers that were 5’ end labeled with gamma ATP 32P. Radiolabelled riboprobe and oligo probes were hybridized to nitrocellulose membranes pre-blocked with hybridization solution in riboprobes hybridization solution (50% formamide, 5X SSC, 1X Denhardt’s solution, 0.5mg/ml fish sperm DNA, 10mM EDTA, 0.2% SDS) at 65 oC or oligo probes hybridization solution (6X SSC, 10X Denhardt’s solution, 0.1% SDS) at 42 oC respectively. Riboprobed blots were washed twice at room temperature in 2XSSC/0.1%SDS for 5 minutes each wash then for 1 hour at 65 oC in 0.1XSSC.0.1%SDS. Oligo probed blots were washed twice with 6XSSC/0.1%SDS for 15 minutes at room temperature then once at 50 oC in 6XSSC/0.1%SDS for 15 minutes. Probed blots were subjected to phosphorimager analysis and probe intensities were determined using image quant software. For quantitative RT-PCR total RNA was treated with DNase I (Roche) to remove residual genomic DNA contamination. cDNA was created using gene specific primers and reverse transcription with Superscript II (Invitrogen). Reverse transcription reactions were set up according to the manufacturers suggestions with 1ug of DNase I treated total RNA and minus enzyme controls were included to insure reverse transcription dependent downstream amplification. 1ul of the reverse transcription reaction was used as template for quantitative polymerase chain (qPCR) reactions using the VeriQuestTM SYBR® Green qPCR kit (USB). Chromatin Immunoprecipitation Assays Chromatin immunoprecipation assays (ChIP) were done as previously described with a few alterations (Houseley et al., 2008). 200 mL cultures of yeast strains yJC151 (WT) and yJC327 (dcp2Δ) were grown in synthetic complete media with 2% raffinose as the only sugar source until an OD600 of 0.35. At which time the sugar source was switched to 4% galactose and aliquots at 0, 30, 180, and 360 minutes after galactose addition were crosslinked with formaldehyde at a final concentration of 0.25%. Cultures were crosslinked for 5 minutes after which glycine was added to a final concentration of (140mM). Analogous cultures were grown in parallel for RNA analysis where crosslinking was omitted. RNA analysis was performed as described above. After crosslinking the cultures the cells were kept cold on ice. The cell pellets were collected

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by centrifugation at 4,000rpm at 4 oC, for 2 minutes, and washed with 10ml ice cold TBS. Cell pellets were transferred to 2ml eppendorf tubes with 1ml TBS and then frozen and stored at -80 oC until lysis. Cell pellets were resuspended in 400ul of lysis buffer (0.1% Deoxycholic acid, 1mM EDTA, 50mM HEPES/KOH, pH7.5, 140mM NaCl) and equal volume glass beads were added. Pellets were bead bashed in the cold room (4 oC) by vortexing for 2 minutes followed by 3 minutes on ice repeated 5 times. The lysate was separated from the beads by puncturing a whole in the 2ml tube followed by centrifugation at 2,000rpm for 2 minutes at 4 oC. The insoluable pellet was brought into solution by vortexing and then triton was added to the lysis buffer to a final concentration of 1%. The slurry was micrococcal nuclease treated with 75 Units of micrococcal nuclease and CaCl was added (2mM final) to start the reaction which was incubated for 5 minutes at 37 oC. The reaction was stopped by the addition of EGTA to a final concentration of 5mM. 20OD units of lysate was aliquoted into 1.5ml tubes and brought up to a final volume of 600ul. Then the 5ul of ChIP grade antibody recognizing acetyl-Histone H3 Lys18 (Millipore Catalogue No. 07-354) were incubated with the lysate overnight with gentle mixing on a nutator at 4 oC. The lysate/antibody mix was then added to 50ul of Protein G Dynabeads (Invitrogen) that had been equilibriated with lysis buffer. After two hours of mixing on a nutator at 4 oC the beads were pulled down and washed successively with 200ul of lysis buffer (0.1% Deoxycholic acid, 1mM EDTA, 50mM HEPES/KOH, pH7.5, 140mM NaCl, 1% Triton) then lysis buffer 500 (0.1% Deoxycholic acid, 1mM EDTA, 50mM HEPES/KOH, pH7.5, 500mM NaCl, 1% Triton), then LiCl/detergent wash (0.5% Deoxycholic acid, 1mM EDTA, 250mM LiCl, 0.5% NP-50, 10mM Tris-HCl, pH8) and then TBS. The beads were transferred to a fresh tube with the TBS wash. The immunoprecipitated material was eluted by incubation at 95 oC for 5 minutes in 100ul of 1%SDS/1XTE. Then the beads were washed with 150ul of 0.67%SDS/1XTE that was then added and the crosslinking was reversed by incubation at 65 oC for 10 hours. After which a proteinase K treatment was done by adding 20ul of Proteinase K 10mg/ml and 230ul of TE and incubating for 2 hours at 37 oC. Nucleic acids were isolated by adding 55ul of 4M LiCl and extraction with Phenol/Chloroform/LET followed by ethanol precipitation with glycogen. qPCR with the primer sets in Sup. Table 6 was then done to quantitative measure the amount of immunoprecipitated DNA. Western Analysis Yeast cells were grown to mid-log phase in synthetic media with 2% glucose then harvested. Cell pellets were resuspended in 5M Urea, then lysed cells with glass beads. A solution of 125 mM Tris, pH 6.8 and 2% SDS was added and the lysis was continued. The debris was then pelleted and the supernatant retained. The gel was loaded with 1 OD unit of protein with SDS, after being denatured at 95 oC for 5 minutes. After running the protein was transferred to membrane by electrophoresis. The membrane was blocked with milk and then probed with an antibody recognizing influenza virus hemagglutinin (HA) (Covance catalog no. MMS-101P). A rabbit anti-RPL5 antibody, obtained from Dr. John Warner, was used as a loading control.

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Figure S1. Deadenylation by CCR4, the Decapping Activators DHH1 and LSM1, and the Decapping Enzyme DCP2 Are Required for Efficient mRNA Decay, Related to Figure 2 (A-E) Cells transformed with a reporter MFA2 mRNA under the control of the GAL1 upstream activating sequence were grown to mid-log phase in the presence of 2% galactose and 1% sucrose to activate transcription of the reporter. Transcription of the reporter was turned off by changing the growth media to 4% glucose, aliquots were taken and RNA was analyzed by Northern analysis. RNA levels were normalized to the SCR1 RNA loading control and half-lives were determined for (A) WT, (B) ccr4Δ, (C) dhh1Δ, (D) lsm1Δ, and (E) dcp2Δ cells.

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Figure S2. GAL10 lncRNA Degradation Is Dependent on the Catalytic Activity of DCP2, Related to Figure 2 To validate that GAL10 lncRNA turnover requires DCP2 and that the observed stability of this lncRNA is not a consequence of a secondary mutation that could have arisen in our dcp2Δ yeast strain, we performed complementation analysis. Specifically, we measured GAL10 lncRNA half-lives in dcp2Δ cells complemented with either a wild-type DCP2 gene or an allele harboring a mutation within the NUDIX domain that results in loss of in vivo decapping activity (i.e. dcp2-4; Dunckley and Parker, 1999). (A) Equivalent levels of DCP2-HA and dcp2-4-HA protein expression in dcp2Δ cells were determined by Western Blot analysis. Specifically, dcp2Δ cells were transformed with a

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centromeric plasmid expressing either an HA-tagged wild-type copy of DCP2 or the dcp2-4 allele. As a control dcp2Δ cells were also transformed with an empty vector. Cells were grown into mid-log phase and cell lysates were run out on SDS PAGE gel for immunodetection with an antibody against the HA epitope tag. Endogenous RPL5 was used as a loading control. (B) Half-life analysis of GAL10 lncRNA demonstrated that wild-type DCP2 fully complemented the dcp2Δ mutation while the loss-of-function dcp2-4 allele failed to restore wild-type rates of GAL10 lncRNA turnover. Cells were grown in the presence of raffinose until mid-log phase and then were shifted to galactose. GAL10 lncRNA half-lives were determined for WT transformed with an empty vector, dcp2Δ cells transformed with a plasmid expressing a wild-type copy of DCP2, dcp2Δ cells transformed with an empty vector, or dcp2Δ cells transformed with a plasmid expressiong the dcp2-4 allele. SCR1 RNA serves as the loading control. (C) Quantification of band intensities of (B) were normalized to SCR1 RNA. GAL10 lncRNA levels are represented as a percentage of the WT 0 min time point.

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Figure S3. Decapping Enzyme Mutant Cells Display a Strong Slow Growth Phenotype in the Presence of Galactose, Related to Figure 4 Equivalent cell numbers of WT and dcp2Δ cells were spotted as serial dilutions on solid media with either glucose or galactose as the sole sugar source.

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Table S1. Previously Characterized S. cerevisiae lncRNAs Identified as DPC2 Substrates, Related to Figure 1

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Table S2. lncRNA, Inannotated, and Antisense DCP2 Substrates, Related to Figure 1

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Table S3. Genes Proximal to DCP2-Degraded lncRNAs Can Be Grouped into Biological Processes, Related to Figure 7

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Table S4. Yeast Strains, Related to the Experimental Procedures

Table S5. Plasmids, Related to the Experimental Procedures

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Table S6. Oligonucleotides, Related to the Experimental Procedures

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