No abstract

RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.

Most studies about serpin peptidase inhibitor, clade A member 3 (SERPINA3) has been limited to its inhibitory functions and mechanisms. Herein, we report a novel role of SERPINA3 in transcriptional regulation of HCC progression-related genes. Among 19 selected genes through HCC cell isolation system based on telomere length, microarray analyses, and cell-based studies, SERPINA3 was the strongest determinant of increases in telomere length, HCC cell proliferation, survival, migration, and invasion. We also found that SERPINA3 strongly interacted with heterogeneous nuclear ribonucleoprotein K (HNRNP-K) under H 2 O 2 exposure, and the oxidation-elicited SERPINA3-HNRNP-K complex enhanced the promoter activities and transcript levels of a telomere-relating gene ( POT1 ) and HCC-promoting genes ( UHRF1 and HIST2H2BE ). Intriguingly, the inhibition of SERPINA3 oxidation rendered the transcriptional activity of the SERPINA3-HNRNP-K complex suppressed. Moreover, the co-immunoprecipitated HNRNP-K with SERPINA3 quantitatively correlated with not only the level of SERPINA3 oxidation but also the level of POT1 , UHRF1, and HIST2H2BE transcripts and telomere length in HCC tissues. Therefore, the upregulated transcriptional activity of HNRNP-K mediated by SERPINA3 promotes HCC cell survival and proliferation and could be an indicator of poor prognosis for HCC patients.