Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis

Fanconi Anaemia (FA) is a rare human genetic disease characterized by bone marrow failure, malformations, chromosomal instability and cancer susceptibility. Thirteen genes belonging to a common pathway have been identified, but their function is still unclear even if evidence indicates a role in DNA-repair. In the attempt to gain new insights on FA-BRCA pathway, this work aimed at finding and characterizing novel putative interactors of the FA proteins. Using the yeast two-hybrid system, we screened a cDNA library of human testis and rescued two clones. Clone 54, which encoded for a putative ubiquitin-conjugating enzyme E2 (UBE2U), was first found to interact with FANCD2 and then with FANCL (E3 ubiquitin-ligase of the FA pathway), FANCC, FANCE and FANCF by direct interaction mating in yeast. Different assays indicated that the expression of this gene is limited to mouse and human testis (specifically in spermatocytes and spermatides). Interestingly, even mouse Fancd2 showed a high expression level in these two cell types, supporting the hypothesis of an interaction between the two proteins and a role of the FA-BRCA pathway during spermatogenesis. In order to confirm the binding between UBE2U and FANCD2, we transiently transfected cell lines with a tagged UBE2U. However, since we failed to detect the protein at any level, we tried to validate the interaction using mouse testis extract. Using the specific antibody we generated, we were however not able to confirm the binding, but, before excluding definitively the interaction, we should further investigate using more suitable antibodies. Clone 4, encoding for a novel putative exonuclease (ISG20L2), was instead found to interact with the C-terminus of FANCG. Though it was ubiquitously present at low levels in all the cells tested, it showed a stronger expression in mouse testis. In transiently transfected cells, ISG20L2 was detected primarily in nucleoli by immunofluorescence, but it was revealed also in the cytoplasmic fraction by western blot. Both nuclear and cytoplasmic distributions of the protein were confirmed at endogenous levels, after production of a specific antibody. Coimmunoprecipitation studies between ISG20L2 and FANCG did not confirm their interaction, but this might be in agreement with a recent report for ISG20L2 as a nucleolar exoribonuclease, not directly involved with DNArepair.