title: Structural determinants of Arabidopsis thaliana Hyponastic leaves 1 function in vivo.
abstract: MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Paula
authors.initials: P
authors.lastname: Burdisso
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina.
authors.forename: Fernando
authors.initials: F
authors.lastname: Milia
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; Center for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB-UB), Edifici CRAG-Campus UAB, Bellaterra (Cerdanyola del Vallés), Barcelona, Spain.
authors.forename: Arnaldo L
authors.initials: AL
authors.lastname: Schapire
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
authors.forename: Nicolás G
authors.initials: NG
authors.lastname: Bologna
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina.
authors.forename: Javier F
authors.initials: JF
authors.lastname: Palatnik
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: Rasia
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D002942:Circular Dichroism; D018506:Gene Expression Regulation, Plant; D035683:MicroRNAs / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D018749:RNA, Plant / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; C420966:HYL1 protein, Arabidopsis; D035683:MicroRNAs; D018749:RNA, Plant; D016601:RNA-Binding Proteins; C402383:DCL1 protein, Arabidopsis; D043244:Ribonuclease III