Kinasemax

Epitope-tagged proteins were purified using anti-FLAG resin as described above, then eluted with 3xFLAG peptide (Sigma). Protein-associated RNA was isolated by phenol-chloroform extraction as described previously (11 (link)), then treated with DNaseI (DNA-free, Ambion). To detect all RNA species, the protein-associated RNA was dephosphorylated using Calf Intestine Alkaline Phosphatase, then 5′ end radiolabeled using T4 polynucleotide kinase (KinaseMax, Ambion). Labeled RNA was resolved alongside a single-stranded RNA ladder (New England Biolabs) in a 6% polyacrylamide gel with 7 M urea. To quantitatively detect specific transcripts, cDNA was generated from protein-associated RNA according to manufacturer's instructions using SuperScript III reverse transcriptase (Invitrogen) with oligo-dT₂₀N (38 ng/μl) and random 9-mers (10 ng/μl) as primers. The levels of particular transcripts were determined by qPCR and normalized to transcript abundance in the input fraction, as well as to transcript abundance in an immunoprecipitation from a wild-type strain lacking an epitope-tagged protein. Primers used for PCR are listed in Supplementary Table S3.

Total RNA was isolated using TRIzol (Invitrogen) and resolved alongside a single-stranded RNA ladder (New England Biolabs) in a 6% polyacrylamide gel containing 7 M urea. The RNA was transferred to a Hybond-XL nylon membrane (Amersham) and crosslinked by UV irradiation (120 mJ; UV Stratalinker 2400, Stratagene). Crosslinked membranes were blocked with ULTRAhyb-Oligo solution (Ambion) for 30 min at 42°C, then incubated overnight at 47°C with DNA probes complementary to the SRP RNA (Supplementary Table S3), which had been 5′ end radiolabeled according to manufacturer's instructions (KinaseMax, Ambion). Hybridized membranes were washed four times, 30 min each (2x SSC, 0.5% SDS), then imaged using a storage phosphor screen (Amersham).