Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be ...Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm.Here,we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis.We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs.Interestingly,protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain.Moreover,mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37.Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex,which in turn promotes the transcription and proper processing of primary miRNA transcripts(primiRNAs).Taken together,these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes,promoting the transcription and processing of primiRNAs rather than the direct export of processed miRNAs from the nucleus.展开更多
基金This work was supported by grants from the ANPCyT(Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica,Argentina)the HFSP(Human Frontier Science Program)+1 种基金the ICGEB(International Center for Genetic Engineering and Biotechnology)to P.A.M.and from the Max Planck Society to P.A.M.and D.W.P.A.M.and A.L.A.are members of CONICETD.A.C.,A.J.G.,and D.G.are fellows of the same institution.
文摘Post-transcriptional gene silencing mediated by microRNAs(miRNAs)modulates numerous developmental and stress response pathways.For the last two decades,HASTY(HST),the ortholog of human EXPORTIN 5,was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm.Here,we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis.We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs.Interestingly,protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain.Moreover,mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37.Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex,which in turn promotes the transcription and proper processing of primary miRNA transcripts(primiRNAs).Taken together,these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes,promoting the transcription and processing of primiRNAs rather than the direct export of processed miRNAs from the nucleus.
