Pseudouridine modification inhibits muscleblind-like 1 (MBNL1) binding to CCUG repeats and minimally structured RNA through reduced RNA flexibility

Abstract

Myotonic dystrophy type 2 is a genetic neuromuscular disease caused by the expression of expanded CCUG repeat RNAs from the non-coding region of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. These CCUG repeats bind and sequester a family of RNA-binding proteins known as Muscleblind-like 1, 2, and 3 (MBNL1, MBNL2, and MBNL3), and sequestration plays a significant role in pathogenicity. MBNL proteins are alternative splicing regulators that bind to the consensus RNA sequence YGCY (Y = pyrimidine). This consensus sequence is found in the toxic RNAs (CCUG repeats) and in cellular RNA substrates that MBNL proteins have been shown to bind. Replacing the uridine in CCUG repeats with pseudouridine (Ψ) resulted in a modest reduction of MBNL1 binding. Interestingly, Ψ modification of a minimally structured RNA containing YGCY motifs resulted in more robust inhibition of MBNL1 binding. The different levels of inhibition between CCUG repeat and minimally structured RNA binding appear to be due to the ability to modify both pyrimidines in the YGCY motif, which is not possible in the CCUG repeats. Molecular dynamic studies of unmodified and pseudouridylated minimally structured RNAs suggest that reducing the flexibility of the minimally structured RNA leads to reduced binding by MBNL1.