This study aims to systematically reveal the new function and molecular mechanism of 5S rRNA regulating cell process. The biotin-labeled probe was synthesized

and the 5S rRBP was obtained by molecular hybridization. 162 5S rRNA interacting proteins was identified by high sensitivity mass spectrometry and bioinformatics analysis. Protein complex-based gene sets analysis was based on ConsensusPathDB. Protein-protein interaction network was constructed basing on STRING database. The GO database was used for acquiring functional annotations of interactive proteins. KEGG database was used for pathway enrichment analysis. The analysis showed that in addition to interacting with translation-related proteins

5S rRNA also interacts with a large number of proteins from microRNA processing pathways

mRNA splicing pathways and signal pathway regulation pathways

etc. The result indicates that 5S rRNA not only have the function of protein translation regulation

but also participate in the regulation of mRNA splicing and other processes. This study points out a new direction for the study of 5S rRNA function and regulatory mechanism.