An engineered RNA binding protein with improved splicing regulation

Abstract

The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1–2 and ZF3–4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1–2 or ZF3–4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1–2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3–4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

MBNL splicing activity depends on RNA binding site structural context

“…Most recent crystallographic studies report that the second ZF in each tandem specifically binds the YGCY motif, whereas the other ZF may interact with RNA less specifically ( 34 ). In contrast, another in-depth analysis describes ZF1-2 as a domain specifically recognizing the YGCY motif while ZF3-4 acts as a more general RNA binding domain ( 37 ). It could suggest that different YGCY motif arrangement may lead to the formation of MBNL-RNA complexes of distinct splicing capacities.…”

“…However, high affinity binding requires at least 9–10 nucleotides outside the YGCY motif with which ZFs also interact ( 34 ). The ZFs are arranged as two tandems not functionally equivalent for all splicing events ( 35 , 36 ) and which differ in specificity of YGCY recognition ( 37 ). These ZFs are separated by an ∼80 residue linker, which could facilitate binding to separated RNA motifs in cis or in trans ( 38 , 39 ).…”

The potential of engineered eukaryotic RNA‐binding proteins as molecular tools and therapeutics

“…Another example of an engineered RNA splicing factor can be found in the recent study that used a rational design method to replace ZF3‐4 of MBNL1 with another copy of ZF1‐2 or ZF1‐2 with a ZF3‐4 domain (Hale et al, ). The former protein with two ZF1‐2's showed a fivefold increase in activity compared to wild type MBNL1, and the latter protein with two ZF3‐4 domains had a fourfold decrease in activity (Hale et al, ). The double ZF1‐2 protein also showed rescue of MBNL1 regulated alternative splicing events in a DM1 disease model (Hale et al, ).…”

“…The former protein with two ZF1‐2's showed a fivefold increase in activity compared to wild type MBNL1, and the latter protein with two ZF3‐4 domains had a fourfold decrease in activity (Hale et al, ). The double ZF1‐2 protein also showed rescue of MBNL1 regulated alternative splicing events in a DM1 disease model (Hale et al, ). While this approach to engineering an RNA splicing factor produced some promising results, it requires an intimate knowledge of a protein's domain structure and functions, limiting its usefulness in the design of other splicing factors.…”

“…Building upon this concept, the 2018 study used a rational design method to replace ZF3-4 with a second ZF1-2 domain in one protein and in another protein replaced ZF1-2 with a ZF3-4 domain. This design strategy allowed the authors to determine the activity of the respective domains (discussed section 4.1) (Hale et al, 2018). That ZFs of this class can be added or deleted from RBPs to modulate activity has implications for the engineering of other CCCH-ZF containing proteins.…”