Structural features defining the in vivo function of A. thaliana HYL1

BURDISSO P; MILIA FJ; SCHAPIRE A; BOLOGNA NG; PALATNIK JF; RASIA RM

Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

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 DCL11 and participates in miRNA strand selection2. 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 interaction3. 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.