Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA binding protein that regulates the alternative splicing of a variety of transcripts. In Myotonic Dystrophy (DM) aberrant cellular localization of MBNL1 results in disease-associated mis-splicing of several MBNL1 target pre-mRNAs. Due to its role in DM pathogenesis, MBNL1 has been a topic of intense study for the last decade, however many open mechanistic questions remain regarding how MBNL1 recognizes RNA substrates to mediate splicing. The RNA recognition motif for MBNL1, 5'-YGCY-3', was defined herein. This motif was used to identify novel MBNL1 binding sites within regulated transcripts and create synthetic MBNL1-regulated splicing reporters. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged into two pairs of two ZFs. A comprehensive, combinatorial mutagenic study of MBNL1 was conducted to determine the role of each ZF in RNA binding and splicing activity. Functional analysis of the mutant proteins in cellular splicing assays and assessment of RNA binding activity demonstrated that the ZF pairs (i.e. ZF1-2 or ZF3-4) do not have equivalent activity. The ZF1-2 pair is responsible for MBNL1's high affinity RNA binding and splicing activity, whereas the ZF3-4 pair has reduced affinity for RNA and impaired ability to regulate splicing of some transcripts. Hierarchical clustering analysis revealed that two distinct classes of MBNL1-regulated splicing events exist within the small set of splicing events examined. For Class II splicing events the binding and splicing activity for the ZF mutants correlated well. However, for Class I events there was no significant correlation between RNA binding and splicing activity. For pre-mRNAs in the latter class it appears that MBNL1 exerts surprisingly robust splicing activity in the absence of strong RNA binding, suggesting that MBNL1 may be recruited to some pre-mRNA substrates through protein-protein interactions. This study provides the first demonstration that functionally distinct classes of MBNL1-mediated splicing events exist in terms of requirements for different ZFs and the importance of RNA binding. This dissertation includes previously published and unpublished co-authored material as well as recently co-authored material that has been submitted for publication.