Hasty: from exportin to a miRNA biogenesis factor

LEI L.; ARCE A.L.; MANAVELLA, P.A.; CAMBIAGNO D.A.; WEIGEL D.

Puerto Varas

Congreso; II Molecular Biosystems Conference - Eukaryotic Gene Regulation and Functional Genomics; 2019

Micro RNAs (miRNAs) are 21 nucleotide molecules generated from primary miRNAs (pri-miRNAs), which are transcribed by RNA polymerase II. MIRNA transcription involves the Mediator and Elongator complexes, and Elongator is also important for the recruitment of the processing machinery. SERRATE (SE), HYPONASTIC LEAVES 1 (HYL1) and DICER LIKE 1 (DCL1) are the core components of this processing complex. In plants, miRNA biogenesis is carried out in the nucleus and then exported to cytoplasm to fulfill their functions. Different form humans where EXPORTIN 5 (XPO5) export pre-miRNA to the cytoplasm, in plant is less clear how this process occurs. HASTY (HST), the plant homologue to XPO5, was long proposed to be the protein responsible of the nuclear/cytoplasm movement of miRNAs in plants. However, even when hst mutant plants accumulate less miRNAs, the evidence is not supporting its role as an exporter since the nuclear/cytoplasm partition of miRNA is unaffected in these mutants. Here, we explored the role of HST in the miRNA biogenesis in Arabidopsis thaliana. We corroborated that nuclear miRNA accumulation in hst mutants is not altered, suggesting a HST-independent exportation of miRNAs. We found that HST is predominantly localized in the nuclei, and that such sub-cellular localization depends on RAS-RELATED NUCLEAR PROTEIN-1 (RAN-1), suggesting that it could act shuttling other cargos rather than miRNAs. Coincidently with a potential role in miRNA processing we observed an over accumulation of pri-miRNAs in hst. Interestingly, HST co-immunoprecipitation followed by LC-MS/MS and BiFC assays revealed that this protein interacts with DCL1 and several Mediator subunits by its N- and C-terminal domain respectively. Moreover, TriFC assay revealed a light or no interaction between DCL1 and Mediator, but their interaction is strongly stabilizes by HST, suggesting that this protein act as scaffold to recruit DCL1 to MIRNA genes. Such scenario was confirmed by DCL1-ChIP-qPCR assays in hst mutants where we demonstrated that DCL1 recruitment to MIRNA genes depends on HST. Based on our results, we proposed that HST is required for the interaction between DCL1 and Mediator at MIR genes allowing the recruitment of the miRNA processing machinery to nascent pri-miRNAs.