Edited by Ivan SadowskiKeywords: Gpn1 Gpn3 Gpn1-Gpn3 interaction Gpn1-Gpn3 nucleocytoplasmic shuttling Interdependent protein levels shRNA a b s t r a c t Gpn1 and Gpn3 are GTPases individually required for nuclear targeting of RNA polymerase II. Here we show that whereas Gpn3-EYFP distributed between the cytoplasm and cell nucleus, it was mainly cytoplasmic when coexpressed with Gpn1-Flag. Gpn3-Flag retained Gpn1-EYFP in the cytoplasm. However, Gpn3-EYFP/Gpn1-Flag nucleocytoplasmic shuttling was revealed after inhibiting nuclear export with leptomycin B. All Gpn3-EYFP coimmunoprecipitated with Gpn1-Flag, and all Gpn1-EYFP with Gpn3-Flag. Importantly, most endogenous Gpn1 and Gpn3 also associate. Gpn1-Gpn3 interaction was essential to maintain steady-state protein levels of both GTPases. We propose that most Gpn1 and Gpn3 associate, are mobilized, and function as a protein complex. Structured summary of protein interactions:GPN3 physically interacts with GPN1 by anti tag coimmunoprecipitation (1, 2) GPN3 and GPN1 colocalize by fluorescence microscopy (View interaction)

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.

Abbreviations: Retinoblastoma protein/gene (RB/RB1); External Beam Radiotherapy (EBRT); intravenous chemotherapy (IVC); vincristine, etoposide and carboplatin (VEC); intra-arterial chemotherapy (IAC); intraocular classification of retinoblastoma (ICRB); Proton beam radiation therapy (PBRT); the transforming growth factor b1 (TGF-b1); senescence-associated heterochromatic foci (SAHF); lactate dehydrogenase (LDH); no-homologous end joining (NHEJ); homologous recombination (HR); methyltransferase 1 (DNMT1); DNA-dependent protein kinase (DNA-K); histone deacetylase 1 (HDAC1); DNA-dependent protein kinase (DNA-K); Sex-determining Y box 2 (SOX2); Ras association domain family (RASSF); Cellular Retinoic Acid Binding Protein (CRABP); apolipoprotein A-I (APOA1); Colon Cancer Associated Transcript 1 (CCAT1); glial fibrillary acidic protein (GFAP); Retinol Binding Protein 3 (RBP3); lamin B1 protein(LMNB1); Transferrin Receptor (TFRC).

The retinoblastoma tumour suppressor protein (RB) regulates a number of diverse cellular functions including differentiation, angiogenesis, chromatin remodelling, senescence and apoptosis. The best-characterised function of RB is cell cycle regulation, and it has been considered a phosphoprotein regulated by cyclindependent kinases. In its hypophosphorylated form, RB binds the transcription factor E2F1, arresting the cell cycle in the G1 phase. Here, we show that MDM2 controls the cell cycle through synthesis and degradation of RB protein in a cell cycle condition-dependent fashion. MDM2 induces G1 cell cycle arrest by enhancing the translation of the RB mRNA under genotoxic stress. Translation requires direct interaction between the RB mRNA and the MDM2 protein that accompanies the RB mRNA to the polysomes. However, MDM2 ubiquitinates and degrades RB protein at the G2/ M phase under genotoxic stress. The ATM phosphomimetic mutant MDM2(S395D) corroborates that the effect on the RB levels is dependent on the DNA damage. These results provide the basis of a dual regulatory mechanism by which MDM2 controls cell cycle progression during DNA damage.

Retinoblastoma is a pediatric neoplasia with a high incidence in non-developed countries. Nowadays the diagnosis is clinical and unfortunately in advanced stages of the diseases, which puts the child´s life risk. The search for molecular diagnosis on retinoblastoma is necessary. Due to their location and tendency to migrate, biopsies of retinoblastoma are not recommended then, peripheral blood samples can be a good source for the search of biomarker. RT qPCR is a sensitive method for gene expression quantification; in order to achieve optimal results, a crucial step is the reference gene choice that cannot be neglected under any circumstance. Six of the most commonly used housekeeping genes; GAPDH, HPRT1, B2M, TBP, RPL13a and 18S, were tested in the blood samples of patients diagnosed with retinoblastoma and healthy controls.The HPRT and TBP were found the most reliable genes whereas GAPDH, that is one of the most commonly used genes for normalisation, together with B2M, RPL13a and 18S have to be avoided. Using the selected reference genes, the Rb mRNA showed significant differences between patients and healthy children whereas no differences were found using to control groups. In the present study, we validate blood samples of patients with retinoblastoma.