Recombinant rnasin ribonuclease inhibitor

Experiments were performed as previously described^{22 (link)} with minor modifications. Strains were grown at 28 °C in 250 mL of YPD media to OD₆₀₀ = 1.0. Extracts were lysed in TMG (10 mM Tris-HCl, pH 8.0, 1 mM MgCl₂, 10% glycerol) plus 200 mM NaCl, PMSF (1:10 dilution), Set III Protease inhibitor cocktail (Calbiochem, 1:10 dilution), and 30 μL Recombinant RNasin Ribonuclease Inhibitor (Promega). Ten milligrams of protein was incubated with 4 μL α-myc antibody (Sigma M4439) for 1 hour at 4 °C, 100 μL of Pierce™ Protein G magnetic beads (Thermo Fisher Scientific 88847) was added and rotation continued for 2.5 hours. Following bead washing as previously described^{22 (link)}, 10 μL of IP and 70 μg of input were reserved for western blotting. The remaining IP and input reactions were incubated at 37 °C for 30 minutes in Proteinase K solution (2 mg Proteinase K, 0.01 M Tris-HCL, 0.1 M NaCl, 1% SDS, 0.01 M EDTA). RNA extraction and subsequent northern blot analysis was performed as described below. To monitor pull-down efficiency, reserve IP and input samples were analyzed by SDS-PAGE on a 10% gel, transferred to an Immobilon-FL PVDF membrane (Millipore), and probed with α-myc antibody (Sigma C3956, 1:1,000). TLC1 RNA levels and Est1 pull-down were quantified using ImageQuant software (GE Healthcare Life Sciences).

For semi-quantitative PCR (qPCR), total RNA was prepared by using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The cDNA synthesis was carried out with 200 ng of RNA by using 5U RevertAid™ H Minus Reverse Transcriptase (Fisher Scientific, Schwerte, Germany), 20U Recombinant RNasin® Ribonuclease Inhibitor (Promega, Mannheim, Germany), 0.5 μl 28 μmol polyd(T)₁₂₋₁₈ Primer (Carl Roth, Karlsruhe, Germany), 0.1 μg Random Hexamer Primer (Fisher Scientific), 0.2 μg dNTP-Set 1 (Carl Roth). Intron-spanning primers were designed using web-based Primer3 (19 (link)). The cycling program consisted of 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s and 72°C for 30 s on a LightCycler 480 (Roche Applied Science, Mannheim, Germany). The PCR mixture contained 1.25 mM MgCl₂, 200 μM each dNTP, 0.24 μM of each primer (Supplementary Table 2), 1X Evagreen Dye (Biotrend, Köln, Germany) and 0.5 U of BIOTAQ DNA polymerase (Bioline, Luckenwalde, Germany). Gene expression values were determined by using the 2-ΔΔCT method (20 (link)) with actb and rplp0 as reference genes and normalized to the lowest calculated value for each gene.

First-strand cDNA synthesis was performed using recombinant Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega Corporation, Madison, WI, USA) and a blend of random hexamer primers (Roche Diagnostic, Indianapolis, IN, USA). The first phase of the reaction was carried out by incubating 5 μl of total RNA with 0.5 ng/μl of random hexamer primers for 5 min at 70 °C. Samples were kept on ice for 10 min. The final volume of the second phase was 25 μl per reaction including 5 μl of M-MLV 5X reaction buffer, 2.5 mM dNTPs, 25 units of recombinant RNasin ribonuclease inhibitor (Promega Corporation, Madison, WI, USA), and 200 units of M-MLV reverse transcriptase enzyme (Promega Corporation, Madison, WI, USA). Samples were incubated for 1 h at 37 °C and the resulting cDNA was stored at − 20 °C.