Abstract
MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.
Highlights
Following their generation from the DROSHA and DICER complexes [1], mature miRNAs associate with members of the Argonaute (AGO) and TNRC6 families of proteins, leading to the formation of the RNA-induced silencing complex (RISC)
We found that S6K2 and TRBP interacted in untreated human dermal lymphatic endothelial cells (HDLECs) but the interaction was dramatically increased at 12 h post ANG1 addition (Figure 5A, B and Supplementary Figure S5A)
We describe a previously uncharacterized S6K2-mediated mechanism controlling miRNA biogenesis in human primary cells, through regulation of TRBP phosphorylation and expression (Supplementary Figure S7H)
Summary
Following their generation from the DROSHA and DICER complexes [1], mature miRNAs associate with members of the Argonaute (AGO) and TNRC6 (trinucleotide repeat containing 6) families of proteins, leading to the formation of the RNA-induced silencing complex (RISC). MiRNAs play a central role in mammalian development [2] This is evidenced by the fact that genetic deletion of DICER in mice causes vascular abnormalities leading to embryonic lethality [3]. The intracellular signalling mechanisms leading to post-translational modification of the miRNA biogenesis machinery in non-transformed cells, including DROSHA, DGCR8 (DiGeorge syndrome chromosomal region 8), TRBP (TARBP2; transactivating response RNA (HIV-1)-binding protein 2) and AGO2 [8,9,10,11], remain poorly understood. Kim et al identified 10 more potential TRBP phosphorylation sites [19]. Both studies suggested that TRBP hyper-phosphorylation controls its stability [10,19]. The relevance of TRBP post-translational modifications to its function in physiological responses in human primary cells remains poorly understood
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