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Domain I of ribosomal protein L1 is sufficient for specific RNA binding.

ABSTRACT: Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.

SUBMITTER: Tishchenko S

PROVIDER: S-EPMC2175363 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Domain I of ribosomal protein L1 is sufficient for specific RNA binding.

Tishchenko Svetlana S Nikonova Ekaterina E Kljashtorny Vladislav V Kostareva Olga O Nevskaya Natalia N Piendl Wolfgang W Davydova Natalia N Streltsov Victor V Garber Maria M Nikonov Stanislav S

Nucleic acids research 20071025 21

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain ...[more]

PMID: 17962298

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Project description:BackgroundRibosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53.MethodsGene knockdown was achieved in HCT116p53+/+, HCT116p53-/-, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice.ResultsRSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model.ConclusionWe report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development.

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Domain I of ribosomal protein L1 is sufficient for specific RNA binding.

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Domain I of ribosomal protein L1 is sufficient for specific RNA binding.

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