Yeast Two-Hybrid Screening for RPGRIP1L Interactors

YTH screen was performed in Saccharomyces cerevisiae strain L40 (trp1-901, his3D200, leu2–3, ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) using MyoVa-GTD cloned into pBTM116 (LexA DNA-binding domain, DBD) as bait and a human fetal brain cDNA library (Clontech) cloned into pACT2 (Gal4 activation domain, AD) as prey. Yeast cells were transformed with pBTM116_MyoVa-GTD vector and the library as described by Alborghetti and co-workers47 (link). The screen was performed in solid Synthetic Defined Medium without tryptophan, leucine and histidine (SD-WLH) containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich, St. Louis, MO). To identify the preys, the pACT2 plasmids of positive clones were isolated and sequenced. The DNA sequences were then compared with those available in the NCBI data bank using the BLASTX program48 (link). The clone identified as encoding for the full-length RPGRIP1L isoform c (NP_001295263.1) was further selected for in vitro and in cell validation and characterization.