EMSA and RNase H protection assay

EMSA was performed using RNA oligos synthesized by Sigma, Life Science and radio-labelled using [γ-³²P] ATP and T4 polynucleotide kinase (New England Biolabs). Binding reactions containing labelled RNA probes, together with nuclear extracted obtained from transfected HEK-293 cells, were performed in 1 × binding buffer (10 mM NaCl₂, 10 mM Tris pH 8.0, 2 mM MgCl₂, 5% glycerol and 1 mM DTT) for 60 min at room temperature before electrophoresis on a 8% polyacrylamide native gel at 100 V for 1.5 h in 0.5 × Tris borate/EDTA buffer at 4 °C. Super-shifts were obtained by adding antibodies specific to U1A, U170K and U1C. A pre-run of the gel (approximately 20 min) was performed before the samples were loaded. Following electrophoresis, the gels were dried on 3MM Whatman paper and exposed on a Cyclone Phosphor screen (Packard). For RNase H protection assays, whole-cell extracts in RSB-100 buffer were incubated with 5 μM antisense DNA oligonucleotide complementary to U1snRNA (5′-CAGGTAAGTAT-3′) and 1.5 U RNase H (Promega) for 30 min at 30 °C. Total RNA was extracted and analysed by northern blotting on a 12% polyacrylamide gel using an internal U1 probe (5′-CAAATTATGCAGTCGAGTTTCCCACATTTG-3′).