Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis
Year: 2009
Type: dissertation
Abstract: Fanconi Anaemia (FA) is a rare human genetic disease characterized by bone marrow \nfailure, malformations, chromosomal instability and cancer susceptibility. Thirteen genes \nbelonging to a common pathway have been identified, but their function is still unclear even \nif evidence indicates a role in DNA-repair. In the attempt to gain new insights on FA-BRCA \npathway, this work aimed at finding and characterizing novel putative interactors of the FA \nproteins. Using the yeast two-hybrid system, we screened a cDNA library of human testis \nand rescued two clones. Clone 54, which encoded for a putative ubiquitin-conjugating \nenzyme E2 (UBE2U), was first found to interact with FANCD2 and then with FANCL (E3 \nubiquitin-ligase of the FA pathway), FANCC, FANCE and FANCF by direct interaction \nmating in yeast. Different assays indicated that the expression of this gene is limited to \nmouse and human testis (specifically in spermatocytes and spermatides). Interestingly, even \nmouse Fancd2 showed a high expression level in these two cell types, supporting the \nhypothesis of an interaction between the two proteins and a role of the FA-BRCA pathway \nduring spermatogenesis. In order to confirm the binding between UBE2U and FANCD2, we \ntransiently transfected cell lines with a tagged UBE2U. However, since we failed to detect \nthe protein at any level, we tried to validate the interaction using mouse testis extract. Using \nthe specific antibody we generated, we were however not able to confirm the binding, but, \nbefore excluding definitively the interaction, we should further investigate using more \nsuitable antibodies. Clone 4, encoding for a novel putative exonuclease (ISG20L2), was \ninstead found to interact with the C-terminus of FANCG. Though it was ubiquitously present \nat low levels in all the cells tested, it showed a stronger expression in mouse testis. In \ntransiently transfected cells, ISG20L2 was detected primarily in nucleoli by \nimmunofluorescence, but it was revealed also in the cytoplasmic fraction by western blot. \nBoth nuclear and cytoplasmic distributions of the protein were confirmed at endogenous \nlevels, after production of a specific antibody. Coimmunoprecipitation studies between \nISG20L2 and FANCG did not confirm their interaction, but this might be in agreement with \na recent report for ISG20L2 as a nucleolar exoribonuclease, not directly involved with DNArepair.
Author: Fabio Morgese
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1596153710