STRUCTURAL FEATURES DEFINING THE FUNCTION OF THE miRNA PROCESSING PROTEIN HYL1 IN VIVO

BURDISSO, PAULA; MILIA, FERNANDO; SCHAPIRE, ARNALDO; BOLOGNA, NICOLAS G.; PALATNIK, JAVIER F.; RASIA, RODOLFO M.

Mendoza

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Structural features defining the in vivo function of the miRNA processing protein HYL1. Burdisso Paula, Millia Fernando J, Schapire Arnaldo, Bologna Nicolás G, Palatnik Javier F and Rasia Rodolfo M Instituto de Biología Molecular y Celular de Rosario, Facultad de Cs Bioq y Farm. UNR-CONICET. HYL1 is a protein involved in microRNA (miRNA) biogenesis that contains two double-stranded RNA binding domains (dsRBDs). HYL1 enhances the efficiency and precision of the RNase III protein DCL1 and participates in miRNA strand selection. We have previously demonstrated that the first domain (dsRBD1) is the major contributor to RNA binding and we defined three main binding regions (R1, R2 and R3) based on the NMR mapping of the interaction. With the aim of gaining insight on the contribution of each region to HYL1 activity, we constructed five dsRBD1 mutants (one located in R1, three in R2 and one in R3) and we transformed hyl1 plants to evaluate phenotype rescue. We found that regions R1 and R3 are the most relevant for in vivo HYL1 function. These plants have a lower phenotype recovery percentage and RT-qPCR assays showed miRNA and pre-miRNA levels similar those of hyl1 deficient plants. To better understand the underlying reasons of the differential behavior of HYL1 mutants, we further biophysically characterized the mutant domains. The 15N-HSQC spectra show that all mutant domains are well folded and that the low efficiency in processing of R1 mutants may be the result of a decrease in the protein stability whereas R3 mutants may have a lower substrate binding affinity. Our results highlight the multiplicity of factors that determine in vivo protein activity.