Functional assignments of the ubiquitination sites K62 and K230 of the human ribosomal protein uS3 (RPS3)

Ribosomal protein uS3/RPS3 in eukaryotes is an essential component of the 40S ribosomal subunit, where it interacts with mRNA and initiation factors. It also has various extra-ribosomal functions. Proteomic studies have identified several ubiquitination sites in human uS3 that could potentially serve either as degradation signals to limit the protein level or as regulatory signals to target uS3 into cellular pathways. Currently, only uS3 ubiquitination at K214 is well understood. Here, we obtain information on functional assignment of uS3 ubiquitination at K62 and K230, using HEK293T cells producing FLAG-tagged uS3 or its forms with replacements K62R or K230R incapable of ubiquitination. Both mutations lead to a large increase of the total uS3 level in the cell indicating the involvement of the respective sites in a degradatory ubiquitination. A comparison of the level of reporter mRNAs in the total cell lysate and in the polysomal fractions revealed that both K62 and K230 are involved in the regulation of the cellular level of ribosome-free uS3 and their replacement with arginine reduces translation efficiency. Furthermore, free uS3 increases the relative levels of translated mRNA of NF-κB-dependent genes such as IL8, IL23A, and CD40, supporting its involvement in the NF-κB pathway.