Study of interdomain interactions through the use of paramagnetic tags

FLORENCIA C. MASCALI; RODOLFO M. RASIA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

Micro-RNAs (miRNAs) are a class of small endogenous RNAs that negatively regulate the expression of mRNAs through the recognition of complementary sequences. Proteins with double stranded RNA binding domains (dsRBD) are essential in miRNA biogenesis. These proteins recognize pri-miRNA precursors in plants lacking conserved sequences. However, this process is not well characterized. With the aim of understanding the process of recognition of this type of domains, we decided to carry out a structural study of the two dsRBDs domains of HYL1 from Arabidospsis thaliana. The N-terminal portion of HYL 1 containing two dsRBD domains connected by a native loop is sufficient to complement the hyl1 mutant phenotype. We generated constructs with 14 residue lanthanide-binding tags (LBTs) at each end of either domain, and performed PRE (Paramagnetic Relaxation Enhancement) experiments by measuring the signal intensity for each residue in the protein loaded with either Lu(III) as diamagnetic control, or Gd(III), a paramagnetic metal. Unpaired electrons in Gd(III) cause an increase in the relaxation velocity of nearby nuclei as a function of the distance to the metal. Based on the results obtained in each domain, we modeled the dynamics allowed by the loop that connects them. We observed that the movement of the domains is not completely random, and that there is a predisposition for forms connecting the N-terminal end to the second dsRBD domain. Once free system is fully characterized, the complex between these proteins and miRNA precursors will be further studied, in order to better understand how these domains recognize their substrate and thus precisely position the active site of the complex in the precursor.