Post-translational modifications of RINT1 (RAD50-interacting protein 1)

Abstract

Initially found to regulate G2/M cell cycle checkpoint upon irradiation-induced DNA damage, RINT1 (RAD50-interacting protein 1) was later shown to be a multifunctional protein. RINT1 participates in telomerase-independent telomere length maintenance, membrane trafficking between Golgi apparatus and endoplasmic reticulum and ER-Golgi homeostasis. Inactivation of Rint1 leads to an early embryonic lethality. RINT1 can act as tumor suppressor, since heterozygous inactivation results in tumor formation in mice, while in humans mutant RINT1 variants predispose to development of breast- and Lynch syndrome-related cancers. RINT1 was also proposed to be an oncogene for glioblastoma development. Nevertheless, despite its involvement in a variety of biological pathways, no data regarding post-translational modifications (PTM) of RINT1 or their impact on the protein’s function was reported. The present study shows that RINT1 is subjected to two kinds of post-translational modifications: ubiquitination and, putatively, SUMOylation. It was found that RINT1 is a short-lived protein with a half-life (approx. 40 min) regulated by a proteasomal degradation pathway. RINT1 is ubiquitinated by several ubiquitin linkage types. Since K48-mediated polyubiquitin (polyUb) chains as well as K63- and K29-mediated polyUb chains were detected, functional significance of RINT1 ubiquitination is not limited to proteasomal degradation (K48-mediated polyUb chains) but could also serve to regulate multiple cellular functions of RINT1 (K63- and K29-mediated polyUb chains). Interestingly, RINT1 was also found to interact with lysineless ubiquitin mutant. Thus, monoubiquitination or linear ubiquitination (a less characterized and newly discovered PTM) of RINT1 could also be postulated. Furthermore, analysis of in silico predicted ubiquitination sites of RINT1 by co-immunoprecipitation of mutant versions (truncated mutants and site-directed mutagenesis) revealed that RINT1 is ubiquitinated at different sites within the protein. Two E3 ubiquitin ligases, HUWE1 and RNF20/RNF40E3 complex, were identified by mass spectrometry assay as binding partners mediating ubiquitination of RINT1. The specificity of these interactions was subsequently confirmed by co-immunoprecipitation experiments. Importantly, shRNA-induced down-regulation of HUWE1 and RNF20 or RNF40 protein levels resulted in enhanced RINT1 stability, thus indicated their novel role as regulators of proteasomal degradation of RINT1. Moreover, mass spectrometry analysis and yeast two-hybrid assay identified SUMOylation as another covalent modification of RINT1. First experiments suggested covalent modification of RINT1 by SUMO proteins. In conclusion, the present study demonstrated that human RINT1 is a protein of a short-half life, heavily ubiquitinated at different sites within the protein and via different ubiquitin chain linkage types (K29, K48, K63). It is also modified by a lysineless ubiquitin mutant and potentially SUMOylation. Importantly, RINT1 interacts with HUWE1 and RNF20/40 E3 ubiquitin ligases, which tightly control its cellular levels. This study reveals crucial mechanisms governing homeostatic RINT1 turnover and, in this respect, is the first to address the presence and functionality of RINT1 polyubiquitination.