Unraveling the interactions between MBNL1 protein and its RNA targets

myotonic dystrophy;molecular dynamics;protein-RNA interaction;RNA folding;reaction kinetics

Fu, Yuan

关键词: myotonic dystrophy;    molecular dynamics;    protein-RNA interaction;    RNA folding;    reaction kinetics;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34332/Fu_Yuan.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The muscleblind-like protein 1 (MBNL1) is an RNA binding protein with four conserved zinc fingers that plays an important role in the regulation of alterative splicing. It interacts with pre-mRNA and promotes the inclusion or exclusion of exons to form different mRNA transcripts. Besides its regulatory function in alternative splicing, MBNL1 has also been implicated to interact with CUG and CCUG repeat RNAs that are the causative agents of myotonic dystrophy. As a result of binding to the repeating RNA sequences, MBNL1 is abnormally translocated, which leads to many of the misregulated events in myotonic dystrophy. MBNL1 can bind various RNA substrates, most of which share a stem-loop structure with two GC base pairs and pyrimidine-pyrimidine mismatches. However, MBNL1 has been shown to have a general binding preference for single-strand RNA containing 5’YGCY3’ motifs. In this work, the secondary structural preference of RNA targets for MBNL1 protein is investigated. With gel shift assays, steady-state fluorescence measurements along with circular dichroism measurements, I revealed that MBNL1 alters the structure of helical RNA targets upon binding, which may explain the selectivity of MBNL1 for less structured RNA sites. The stability of the stem-loop RNA secondary structure has been shown to be inversely correlated with the affinities of RNA substrates for MBNL1. Moreover, both the association and dissociation rates of MBNL1-RNA complexes are affected by stem-loop stability. I propose that the binding of MBNL1 to stem-loop RNA destabilize the RNA secondary structure and lead to partial melting of the stem region in the RNA substrates. Consistent with this hypothesis, RNA structure stabilizers were shown to destabilize the interaction between MBNL1 and CUG repeat RNA. In addition to characterization of secondary structural preference of MBNL1, the kinetics of association of the MBNL1 – CUG repeat RNA complex has also been examined. Using stopped-flow fluorescence analysis and surface plasmon resonance, I have shown that multiple phases are present during the course of MBNL1 – CUG repeat RNA association. A multiple mode binding model with negative cooperativity has been proposed based on the kinetic data obtained.