The muscleblind protein family's RNA sequence elements, structural elements and novel binding sites defined through SELEX

xv, 106 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.

Myotonic Dystrophy type I (DM1) is caused by muscleblind protein sequestration to aberrantly expanded CUG repeats. When muscleblind is sequestered it can no longer fulfill its role as an alternative splicing regulator, leading to mis-splicing events in both humans and Drosophila. The muscleblind protein family's RNA binding specificity has been minimally characterized. Only one pre-mRNA target in humans, cardiac troponin T (cTNT), has a known MBNL1 binding site. In order to understand muscleblind's RNA binding specificity and identify a consensus binding motif, systematic evolution of ligands by exponential enrichment (SELEX) was performed on both the Drosophila muscleblind protein, Mbl, and the human ortholog, MBNL1. Drosophila has provided a useful model for studying the disease mechanism of DM1. Studies of Mbl's RNA binding specificity to CUG repeats concluded that replacing the U-U mismatches with different pyrimidine-pyrimidine mismatches was tolerated, but no other mutations were. To understand Mbl's RNA binding specificity, SELEX was performed. After 6 rounds, several sequences were identified that bound with high affinity, all containing the 5'-AGUCU-3' consensus motif. One sequence, SELEX RNA 20 was analyzed further. In addition to the guanosine in the consensus motif of SELEX RNA 20, two other guanosines were shown to be protected by Mbl in a footprinting assay, indicating that Mbl has a strong preference for binding guanosine. Also, two "tail" regions of SELEX RNA 20 were shown to be single stranded and required for binding by Mbl. These results indicate that Mbl is a highly specific RNA binding protein with preference for both single and double stranded guanosine-rich regions. A doped SELEX was performed on MBNL1's binding site from the cTNT pre-mRNA to determine which sequences and structural aspects were important for recognition by MBNL1. Pool 5 RNA sequences bound with high affinity, and the motif 5'-YGCUU-3' was selected. This motif was then used to identify new MBNL1 binding sites in pre-mRNAs regulated by MBNL1, SERCA1 and MBNL1. The identification of this motif and two new MBNL1 sites provide insight into MBNL1-mediated alternative splicing. This dissertation includes both my previously published co-authored material and my unpublished co-authored material.

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