α 32p utp

In vitro cleavage assays were performed as described previously^{56 (link)}. FLAG-tagged wild-type Siwi and the FLAG-tagged Siwi mutants were expressed in BmN4 cells, and the proteins were then immunopurified using Dynabeads Protein G and the anti-DDDDK-tag mAb. The purified proteins were incubated at 27 °C for 17 h with the internally ³²P-labeled substrate RNA (piRNA-4 target)^{56 (link)}, and the reaction products were then analyzed by denaturing urea-PAGE. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in OSCs, and the proteins were then immunopurified using Dynabeads Protein G and the anti-DDDDK-tag mAb. The purified proteins were incubated at 27 °C for 17 h with the internally ³²P-labeled flam target RNA or the 5′ ³²P-labeled mdg1 target RNA, and the reaction products were then analyzed by denaturing urea-PAGE. The flam target RNA was transcribed in vitro with a T7 High Yield Transcription kit (Epicenter), using ³²P-α-UTP (PerkinElmer). The template for the in vitro transcription was prepared by PCR using DNA oligos (Supplementary Table 1). The mdg1 target RNAs were purchased from Integrated DNA Technologies.

Plasmids containing the SELENOP 3’ UTR fragments or mutants were linearized with Not I and transcribed with T7 RNA polymerase in the presence of [32P]-α-UTP (Perkin Elmer). Recombinant GST-PTBP1 was incubated with 20 fmol [32P]-α-UTP labeled fragments. Following incubation, complexes were UV irradiated at 254 nm for 10 min on ice and subsequently treated with 20 μg RNase A for 15 min at 37°C. Samples were resolved by 10% SDS-PAGE, and visualized by phosphorimaging. For the mutants in this study, we replaced the U-rich stretches in the interSECIS with either of the other 3 nucleotides. In addition, we also created a version where most of the U residues in the interSECIS were changed to A (see S1 Fig for sequences).

Multiple-round in vitro transcription assays were performed as published previously (50 (link)). Plasmids pJCDP_O and pJCDP_B1 (Table 1) were used as supercoiled P_O and P_B1 templates. Reactions (50-μl mixtures) were performed in a buffer consisting of 50 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl₂, 0.1 mm bovine serum albumin, 10 mm dithiothreitol (DTT), and 1 mm EDTA. Each DNA template (0.25 nm) of supercoiled plasmids pJCDP_O or pJCDP_B1 was premixed with 30 nm σ⁷⁰-containing E. coli RNA polymerase (1 unit/μl; United States Biochemical Corp.), different amounts of purified MbdR-His₆ protein, and different concentrations of the 3-methylbenzoyl-CoA inducer. For multiple-round assays, transcription was then initiated by adding a mixture of 500 μm (each) ATP, CTP, and GTP, 50 μm UTP, and 2.5 μCi of [α-³²P]UTP (3000 Ci/mmol; PerkinElmer Life Sciences). After incubation for 15 min at 37 °C, the reactions were stopped with an equal volume of a solution containing 50 mm EDTA, 350 mm NaCl, and 0.5 mg/ml carrier tRNA. The mRNA produced was then precipitated with ethanol, dissolved in loading buffer (7 m urea, 1 mm EDTA, 0.6 m glycerol, 0.9 mm bromphenol blue, and 1.1 mm xylene cyanol), electrophoresed on a denaturing 7 m urea, 4% polyacrylamide gel, and visualized by autoradiography.

For A₁₅ substrate, 400 nM protein was incubated with 500 nM RNA, 300 μM adenosinetriphosphate (ATP) and 12 μCi/mmol [α-³²P]-ATP (PerkinElmer) in a buffer containing 20 mM Tris–HCl pH 8.0, 300 mM NaCl and 2 mM DTT, supplied with 2 mM different additives, including EDTA, MgCl₂, MnCl₂, ZnCl₂, CaCl₂, NiCl₂ and FeSO₄. For pre-let-7a substrate, 25, 50, 100 and 200 nM protein were individually incubated with 500 nM RNA, 300 μM UTP and 12 μCi/mmol [α-³²P]-UTP (PerkinElmer) in a buffer containing 20 mM Tris–HCl pH 8.0, 300 mM NaCl, 2 mM MgCl₂ and 2 mM DTT. The reaction mixture was incubated at 37°C for 20 min and terminated by adding 2 × RNA loading dye (NEB). A total of 8 μl sample was loaded onto a 20% (for A₁₅) or 10% (for pre-let-7a) polyacrylamide-7M urea gel, which was exposed to a phosphorimaging plate and visualized with a Typhoon TRIO⁺ Variable Mode Imager (GE Healthcare).

Synthesized DNA templates were used in T7 RNA polymerase transcription reactions carried out with the Ambion T7-Megashortscript kit. [α-32P]UTP (800 Ci/mmol; Perkin–Elmer, Waltham, MA, USA) was included in the reaction to label the transcription products [12 (link),25 (link),26 (link),27 (link),28 (link)]. Transcripts of the correct size were purified by electrophoresis on a 10% (wt/vol) denaturing polyacrylamide gel, followed by elution, phenol extraction, and ethanol precipitation. The same conditions were used for all of the reactions: 25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 100 mM NaCl, and 10% (vol/vol) glycerol, 20 fmol of substrate, and equimolar amounts of tRNA endonuclease (2 μM). The reactions were incubated at 30 °C or 65 °C for 8 min. Aliquots were pooled at 2 min intervals, the reaction was stopped by phenol extraction and the ethanol precipitated [12 (link)]. The products were separated on 10% (wt/vol) denaturing polyacrylamide gels and analyzed on a Molecular Dynamics model Storm 860 PhosphorImager using ImageQuant software, version 4. Local average background was corrected, and the fraction cleaved was calculated by the ratio of cleaved product to the sum of the cleaved product plus uncleaved substrate [25 (link)].