FIG 2.

Protein 4 binds RNA in the Y3H system. (A) Schematic presentation of the Y3H system. A hybrid RNA molecule bridges the LexA-MS2 coat and the Gal4AD-TiLV chimeric proteins, resulting in transcriptional activation (arrow) of a lacZ reporter (modified from reference 27). (B) Filter assay for β-galactosidase activity. Yeast colonies expressing the indicated RNA-protein pairs were tested for β-galactosidase activity using colony lift colorimetric assay. Upper panels show images of sections of the filters with stained yeast colonies. Lower panels show Western blot analyses of the expression of the HA-tagged, TiLV protein-Gal4AD fusions in yeast, probed with anti-HA and anti-actin (loading control) antibodies. All fusion proteins showed their expected MW (approximately 56, 55, 39, and 37 kDa for Gal4AD fused to Protein 4, 6, 7, and 8, respectively). For Protein 8-Gal4AD, the lower band (asterisk) matches the calculated MW. (C) Quantitative β-galactosidase liquid assay. For each indicated RNA-protein pair, three yeast colonies were randomly picked from a plate of yeast transformants, expanded, and assayed for β-galactosidase activity using the colorimetric liquid assay. The dashed red line indicates the average β-galactosidase activity of no RNA control for all tested proteins. **, P ≤ 0.05; ***, P ≤ 0.005 (Student’s t test, accounting for multiple testing using the Bonferroni correction). (D) Filter assay for β-galactosidase activity in different time and temperature settings. Panels show images of yeast colonies, expressing the HIVΨ RNA and the indicated TiLV proteins, grown at the indicated periods and temperatures and stained as in panel B. HIV NC served as a positive control. (E) Timeline and conditions for testing Protein 4 RNA-binding activity in the Y3H filter and liquid assays.