amplification and up to 8 mg of the amplified reference RNA product were covalently labeled with Cy5 or Cy3 NHS monoesters (GE Healthcare Life Sciences), respectively. The combined dye-labeled probes were diluted in a 50 ml solution containing 2.56 SSC, 20 mM HEPES-KOH (pH 7.3), 30 mg of poly(A) RNA (Sigma), and 0.2% SDS. The sample was incubated at 95uC for 5 min then centrifuged at 14,000 g for 5 min before application to microarrays. Custom microarrays were designed and spotted as previously described (Hogan et al., 2010). Further information about the probes used in printing the arrays is available from the Operon Web site (https://www.operon.com/; S. cerevisiae YBOX V1.0) and detailed protocols for microarray experiments can be found on the Brown lab website (http:// cmgm.stanford.edu/pbrown/protocols/index.html). Microarray processing prior to hybridization to poly-lysine and epoxysilane glass slides, hybridization using the MAUI hybridization system (BioMicro), as well as post-hybridization washing was performed as previously described [46,71]. Microarrays were scanned using either an AxonScanner 4200 or 4000B (Molecular Devices) and PMT levels were adjusted to achieve 0.1% pixel saturation. Microarray spots were located and analyzed using Genepix Pro 5.0 or 6.0 software (Molecular Devices) and intensity data was submitted to the Stanford Microarray Database [128] for further analysis. Yeast Strain Construction Strains for Immunopurification. The haploid yeast strain PSY2156/JCY18 (MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3- 11,15 ura3 GAL+ PSI+; [129]) was transformed with a 9Myc klTRP1 C-terminal tagging cassette described previously [44]. The 9x MYC C-terminal tagging sequence was integrated at the endogenous loci to generate JCY19 (myo4:9Myc), JCY31 (smy1:9Myc), JCY36 (kip2:9Myc), JCY37 (dyn1:9Myc), JCY38 (myo3:9Myc), JCY39 (myo5:9Myc), and JCY304 (myo2:9Myc). Candidates were confirmed by western blot of total cell lysates using a polyclonal rabbit anti-C-myc (A-14) antibody (Santa Cruz). C-terminal GFP-tagged protein strains [45], including JCY33 (myo4:GFP), JCY34 (smy1:GFP), JCY41 (kip3:GFP), JCY42 (myo2:GFP), JCY43 (vrp1:GFP), JCY44 (las17:GFP), JCY45 (myo5:GFP), JCY46 (kar3:GFP), JCY49 (myo1:GFP), JCY50 (myo3:GFP), JCY51 (kip2:GFP), JCY57 (kip1:GFP), JCY59 (dyn1:GFP), and JCY60 (kar9:GFP) were obtained from Invitrogen. Strains were confirmed by western blot of total cell lysates using a polyclonal rabbit anti-GFP antibody (Living Colors Full-Length A.v., Clontech). Strain genotypes are listed in Table S1. Strains for Microscopy. We chose to use an MS2 system in which the endogenous transcript is expressed from the native promoter since previous studies have indicated that BBC1, LAS17, and VRP1 are expressed at just a one to three copies per cell [98,99], raising concerns about potential spurious results from overexpression of a chimeric transcript from an exogenous vector. In addition, a number of studies have indicated that localization information is frequently found in the 39-UTR of transcripts [2,3], necessitating a method that would retain the native 39-UTR sequence. Strains expressing ARG3, ASH1, BBC1, LAS17, and VRP1 with 12 MS2 hairpins inserted between the open-reading frame and 39 UTR were constructed as described in Haim- Vilmovsky and Gerst [130]. The loxP-SpHIS5-loxP-MS2L cassette was amplified from pLOXHIS5MS2L using gene- targeting primers (Table S7). The tagging cassette was integrated into the haploid yeast genome of JCY66 (MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3) as described in Thompson et al. [107] and candidates were screened via PCR of genomic Actin Patch mRNA Localization and Dynamics preparations using appropriate primers (Table S7). Successful candidates were transformed with the pSH47 plasmid [131] followed by overnight galactose-induced expression of the Cre recombinase in liquid media. Candidates were screened for the successful recombination of the loxP sites and removal of the selectable SpHIS5 marker by the inability to grow on SC his2 media and by PCR of genomic preparations using appropriate primers (Table S7). The resulting strains were then transformed with the pMS2-CP-GFP(x3) plasmid, allowing methionine- inducible expression of the MS2 coat protein (MS2-CP) fused to three copies of EGFP (MS2-CP-GFPx3) in order to create JCY324 (arg3:MS2), JCY107 (ash1:MS2), JCY233 (bbc1:MS2), JCY178 (las17:MS2), and JCY115 (vrp1:MS2). The C-terminal Sac6:mCherry tagged strain (sac6:mCherry) was constructed by amplifying the mCherry KanMX4 cassette from pBS34, obtained from Dr. Roger Y. Tsien, University of California, San Diego, via the Yeast Resource Center (YRC) at the University of Washington, using primers that include sequences complementary to those flanking the stop codon of SAC6 (Table S7). The mCherry:Kan cassette was then transformed into JCY93, also known as EY0987 ([45]; MATa his3D1 leu2D0 lys2D0 ura3D0), to create JCY230. Candidates were selected on plates containing 400 mg/ml G418 (Sigma-Aldrich) and confirmed by western blot using a polyclonal rabbit anti-red fluorescent protein antibody (Rockland) and microscopy. The sac6:mCherry strain, JCY230, was mated to JCY324 (arg3:MS2), JCY107 (ash1:MS2), JCY233 (bbc1:MS2), JCY178 (las17:MS2) and JCY115 (vrp1:MS2) to create the diploid strains JCY335, JCY255, JCY251, JCY267, and JCY263, respectively. In order to assay mRNA localization to FM4–64 internalizing actin patches, the haploid vrp1:MS2 (JCY115) strain was mated to a wildtype MATa strain (JCY93) to create JCY126. In order to construct diploid homozygous MYO3 (myo3D) strains, two different MYO3 knockout cassettes were used. The myo3D::KanMX4 cassette was PCR amplified from a genomic preparation of the myo3D strain in the S. cerevisiae knockout collection (JCY179; [132]) using primers complementary to sequences several hundred bases upstream and downstream of the disrupted MYO3 locus (Table S7). The myo3D::KanMX4 cassette was transformed into las17:MS2 (JCY178), bbc1:MS2 (JCY233), and arg3:MS2 (JCY324), selected on plates containing 400 mg/ml G418 (Sigma-Aldrich), and confirmed by PCR of genomic preparations to create strains JCY395, JCY396, and JCY411, respectively. The myo3D::hph (hygromycin B resistance) cassette was amplified from pBS35 (Dr. Roger Y. Tsien and the YRC) with primers that were designed to include targeting sequences flanking the MYO3 genomic locus (Table S7). myo3D::hph candidates of the sac6:mCherry strain (JCY230) were selected on plates containing 500 mg/ml hygromycin B (Clontech) and confirmed by PCR of genomic preps to obtain JCY392 (sac6:mCherry myo3D:hph). JCY395, JCY396, and JCY411 were mated to JCY392 to obtain the diploid homozygous MYO3 (myo3D) strains JCY405, JCY406, and JCY420, respectively. Sample Preparation for Microscopy 2D Microscopy. Diploid strains for assaying localization of MS2-GFP labeled mRNAs to Sac6:mCherry-labeled actin patches were grown overnight at 30uC in synthetic complete media lacking histidine (SC his2) and supplemented with 40 mg/ml adenine hemisulfate (ade+) and 200 mg/ml G418 (G418+). Strains were back-diluted into fresh SC his2 ade+ G418+ media and grown until early log-phase, then collected and resuspended in SC his2 ade+ G418+ media lacking methionine (SC his2 met2 ade+ G418+) to induce expression of the MS2-CP-GFPx3 protein. PLoS ONE | www.plosone.org 15 February 2012 | Volume 7 | Issue 2 | e31912