What is the clinical evidence from this study?

Study design: Other. Population: Myotonic dystrophy. Intervention: MBNL1 protein. Primary outcome: RNA binding affinity (apparent Kd).

October 17, 2007Open Access

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

MWM. Bryan WarfThird Wave Systems (United States)JBJ. Andrew BerglundAlbany State University

Key Result

MBNL binds short structured CUG and CCUG repeats and the endogenous pre-mRNA target cardiac troponin T with high affinity, recognizing stem-loops containing pyrimidine mismatches.

Structured PICO

Population

In vitro biochemical assays using recombinant truncated MBNL1(1-260) protein and synthetic RNA constructs, and in vivo splicing assays in HeLa cells.

Intervention

Binding of MBNL to short structured CUG and CCUG repeats and cardiac troponin T (cTNT) pre-mRNA.

Outcome

RNA binding affinity (apparent Kd) and structural specificity.surrogate

MBNL binds both pathogenic CUG/CCUG repeats and its normal endogenous target (cTNT pre-mRNA) as structured stem-loops containing pyrimidine mismatches, providing a structural basis for its sequestration in myotonic dystrophy.

Limitations

The UUCG cap used in short RNA constructs may perturb the structure of RNA in a way that negatively affects MBNL binding.
Further studies are necessary to fully define the specificity of MBNL before predictions can be made to identify binding sites in other MBNL regulated pre-mRNAs.

Abstract

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosine-cytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.

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