PRESUPUESTOS MATERIALES DE LA PRISIÓN PREVENTIVA

Although many studies have implicated the DRG proteins to play a role in translation, differentiation and growth, the exact function of this category of TRAFAC class GTP-binding proteins has been difficult to understand.

Early studies have shown that Drg1 in vitro and in vivo, in both mouse and human (Mahajan et al., 1996, Zhao and Aplan 1998) interacts with the oncogenic T-cell acute lymphoblastic leukemia (Tal1/Scl) protein, a basic helix-loop-helix (bHLH) transcription factor involved in cell growth and differentiation (hematopoietic development). Drg1 and another bHLH factor E47 bound to Tal1 in a mutually exclusive manner and competed to interact with Tal1. Drg1 could also bind to Tal2 and Lyl1 which are Tal1 related proteins also having the bHLH domain. It was speculated that in its GTP bound/unbound form Drg1 could bind to Tal1 or release it, which in turn could then translocate to nucleus and bind to its target genes. Mahajan et. al. also showed that overexpression of Drg1 increased rat

embryonic fibroblast transformation induced by c-myc and ras overexpression, affecting both the onset and size of foci formed. Drg2, as mentioned earlier, was also reported to be downregulated in SV-40 transformed (non-tumorigenic) fibroblasts in comparison to normal fibroblasts by a subtractive cDNA cloning approach (Schenker et al., 1994).

In yet another study, filamentous invasion into agar matrices by C. albicans was attenuated by a Drg1 mutant, whereas it did not affect growth in liquid media or filamentous growth in serum. In the agar, both at 25ºC and 30ºC Drg1 null mutant showed defective growth which could be partially complemented by reintroduction of Drg1 gene at its native locus. These results suggested that Drg1 might be important for filamentous growth specifically in matrix-embedded conditions. The null mutant also was defective in filamentation when embedded in YPS, Spider or low ammonium minimal media at 25ºC and 30ºC. Intravenous injection of the C. albicans Drg1 null mutant into mice was also found to cause delayed lethality which in addition, exhibited reduced invasion into tissues (Chen and Kumamoto, 2006).

Drg1 was also found to interact with Efg1, another bHLH transcription factor using the yeast two-hybrid system. C-terminal tagged Drg1 failed to interact with Efg1 implying that Drg1 might bind to bHLH transcription factors through its C-terminal domain as also suggested for Tal1. Efg1 is a repressor of embedded filamentation in C. albicans and it was suggested that Drg1 might play a role in regulating Efg1 as double deletions of drg1 and

efg1 resulted in hyperfilamentation similar to a single efg1 null mutant. That is to say that

the reduced filamentation observed with the single drg1 null mutant was not observed in the Δdrg1Δefg1 mutant (Chen and Kumamoto, 2006).

In an Ubc9 fusion-dependent SUMOylation system (UFDS), Drg1 showed stimulation-dependent SUMOylation induced by the MEKK1 Map3 kinase (Jakobs et al., 2007). The human protein kinase MPSK1 (Myristoylated and palmitoylated serine/threonine kinase 1) also known Stk16, was identified to be an interaction partner of Drg1 by yeast two hybrid and GST pull-down assays. The interaction was independent of the presence of nonhydrolyzable GDP or GTP and the region was mapped onto the N- terminal 1-65 residues of Drg1. MPSK1 was shown to phosphorylate Drg1 at Thr100 present in the G1 motif (Eswaran et al., 2008). Drg1 was also seen to be present in the PI(4,5)P2 and PI(3,4,5)P3 interactomes of the LIM1215 colonic carcinoma cell line as according to an affinity based assay using phosphoinositides immobilized on beads or incorporated into liposomes followed by nanoRP-HPLC ESI MS/MS analysis (Catimel et

1.6.1 Association of DRG factors to translation

The Drg1/Dfrp1 complex was found to be co-localizing with polysomes while the Drg2/Dfrp2 complex was found mostly in the non-polysomal fractions by sedimentation analyses using mouse liver homogenates (Ishikawa et al., 2009). Plant DRG proteins were also shown to be capable of binding to ribosomes, a process partly inhibited by GTPγS (Nelson et al., 2009). Indirect ribosome-Tma46-Drg1 (Fleischer et al., 2006) and yeast Rbg1-Gir2-Gcn1-ribosome (Wout et al., 2009) associations were also suggested. Consistent with these previously available information, it had recently been shown by our collaborators (Dr. Bertrand Seraphin, IGBMC, France), using gradient fractionation of extracts carrying epitope tagged versions of the proteins, that the yeast Drg1-Dfrp1 complex, namely Rbg1-Tma46, associates with the polysomes of the translation machinery (Daugeron et al., 2011). Upon polysome disruption, the complex associates to the 80S monosomes and further with 40S and 60S ribosomal subunits. Rbg2-Gir2 on the other hand did not appear to associate with translating ribosomes and was suggested to be probably recruited to polysomes by Gcn1 under specific conditions. Gcn1 is a translational regulator involved in general amino acid control which was recently shown to interact with Gir2 (Wout et al., 2009).

Deletion of the two yeast DRG-DFRP complexes alone or in combination does not result in a detectable phenotype, thus making it difficult to understand the function of these factors. Interestingly, Daugeron et al. recently reported also that a triple deletion mutant of Rbg1, Rbg2 (yeast Drg homologs) and Slh1 (Ski2-like helicase 1, a putative RNA helicase) exhibits a strong negative growth phenotype in yeast which was not observed with double deletants. Synthetic growth phenotypes were also observed for other combination of mutations inactivating simultaneously the two yeast DRG-DFRP complexes (namely Rbg1- Tma46, Rbg2-Gir2) and Slh1, demonstrating a functional redundancy of these factors. This observation provided for the first time the possibility to perform functional assays for Rbg proteins. Polysome analyses indicated that translation was impaired in the triple deletion mutant with profiles reminiscent of the pattern observed in translation initiation mutants such as cdc33-1 (eIF4E). The amount of polysomes decreased with a subsequent increase in the 80S peak in the mutant strains compared to the wild type strains. Growth complementation analyses using plasmid-borne epitope-tagged versions of the protein allowed the study of the contribution of some of the known conserved motifs in Rbg1 and Tma46, to the function of this complex. This demonstrated that the Rbg1 GTPase activity and Tma46 zinc fingers were required for its function, mutations in the G1 motif (for e.g.:

Rbg1GFPSVGKN) of Rbg1 and CCCH Zn fingers of Tma46 affecting growth as well as