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Title: Identification of putative interactors of Fanconi anaemia proteins by yeast to hybrid system: characterization of two novel genes highly expressed during spermatogenesis
Authors: Morgese, Fabio
Supervisore/Tutore: Savoia, Anna
Issue Date: 21-Apr-2009
Publisher: Università degli studi di Trieste
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.
metadata.dc.subject.classification: MEDICINA MOLECOLARE
Keywords: fanconi anaemia,yeast two-hybrid system
Type: Doctoral
Language: en
Settore scientifico-disciplinare: BIO/10 BIOCHIMICA
NBN: urn:nbn:it:units-7463
Appears in Collections:Scienze biologiche

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