ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.

5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.

PP2Ac/STRN4 negatively regulates STING-type I IFN signaling in tumor-associated macrophages.

Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.

SCMBYK: prediction and characterization of bacterial tyrosine-kinases based on propensity scores of dipeptides.

BACKGROUND: Bacterial tyrosine-kinases (BY-kinases), which play an important role in numerous cellular processes, are characterized as a separate class of enzymes and share no structural similarity with their eukaryotic counterparts. However, in silico methods for predicting BY-kinases have not been developed yet. Since these enzymes are involved in key regulatory processes, and are promising targets for anti-bacterial drug design, it is desirable to develop a simple and easily interpretable predictor to gain new insights into bacterial tyrosine phosphorylation. This study proposes a novel SCMBYK method for predicting and characterizing BY-kinases. RESULTS: A dataset consisting of 797 BY-kinases and 783 non-BY-kinases was established to design the SCMBYK predictor, which achieved training and test accuracies of 97.55 and 96.73%, respectively. Furthermore, the leave-one-phylum-out method was used to predict specific bacterial phyla hosts of target sequences, gaining 97.39% average test accuracy. After analyzing SCMBYK-derived propensity scores, four characteristics of BY-kinases were determined: 1) BY-kinases tend to be composed of α-helices; 2) the amino-acid content of extracellular regions of BY-kinases is expected to be dominated by residues such as Val, Ile, Phe and Tyr; 3) BY-kinases structurally resemble nuclear proteins; 4) different domains play different roles in triggering BY-kinase activity. CONCLUSIONS: The SCMBYK predictor is an effective method for identification of possible BY-kinases. Furthermore, it can be used as a part of a novel drug repurposing method, which recognizes putative BY-kinases and matches them to approved drugs. Among other results, our analysis revealed that azathioprine could suppress the virulence of M. tuberculosis, and thus be considered as a potential antibiotic for tuberculosis treatment.