Expansion beyond a threshold level of CTG repeats in the 3’ UTR of DMPK leads to expression of toxic RNA that, in turn, sequesters MBNL producing the missplicing that underlies disease symptomatology. Knowledge of how the RNA binding protein, MBNL, interacts with RNA to regulate alternative splicing has implications for basic molecular biology as well as therapy development for myotonic dystrophy type 1 (DM1).
Interaction of MBNL with its Regulated Transcripts
Earlier crystallography and targeted mutagenesis studies of zinc finger (ZF) motifs in MBNL1 established their role in regulation of RNA binding and splicing activity (Teplova and Patel, 2008; Purcell et al., 2012). Specifically, four ZF motifs of MBNL1 form two tandem binding domains (ZF1-2 and ZF3-4) that bind YGCY RNA sequences and mediate interaction and posttranslational regulation of target transcripts. While it is established that ZF1-2 and ZF3-4 are not functionally equivalent, their respective roles in RNA binding and regulation are not yet clear.
Assessment of the Roles of ZF Motifs Using Synthetic MBNL1
Dr. Andy Berglund and colleagues (Hale et al., 2018) at the University of Florida have assessed the roles of the ZF tandem binding domains using MBNL protein engineered to express two copies of either ZF1-2 or ZF3-4. Their data show that ZF3-4 acts as a general RNA binding motif (i.e., with less sequence specificity) but one that is relatively less effective in regulating alternative splicing (4x decreased activity over native MBNL). By contrast, MBNL containing duplicate ZF1-2 motifs specifically recognized YGCY motifs and exhibited 5x greater splicing activity than native MBNL. The research team also showed that differences in splicing activity of the two synthetic MBNLs were due to differences in RNA binding specificity, as opposed to binding affinity. There did not appear to be any interdependence of ZF1-2 and ZF3-4. Their data also showed that MBNL with duplicate ZF1-2 motifs could rescue splicing in DM1 cells at a lower concentration than native MBNL.
What Synthetic Proteins Say About MBNL Function and Potential Therapeutics
Structural analyses of ZF motifs have advanced knowledge of how MBNL protein binds RNA and regulates alternative splicing. This understanding of how ZF motifs influence RNA interactions and expression of alternative transcripts may aid development and optimization of MBNL overexpression strategies as a candidate therapeutic for DM1.
Combinatorial mutagenesis of MBNL1 zinc fingers elucidates distinct classes of regulatory events.
Purcell J, Oddo JC, Wang ET, Berglund JA.
Mol Cell Biol. 2012 Oct;32(20):4155-67. Epub 2012 Aug 13.
Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1.
Teplova M, Patel DJ.
Nat Struct Mol Biol. 2008 Dec;15(12):1343-51. doi: 10.1038/nsmb.1519. Epub 2008 Nov 30.
An engineered RNA binding protein with improved splicing regulation.
Hale MA, Richardson JI, Day RC, McConnell OL, Arboleda J, Wang ET, Berglund JA.
Nucleic Acids Res. 2018 Jan 4. doi: 10.1093/nar/gkx1304. [Epub ahead of print]