J. Andrew Berglund

Pentamidine reverses the splicing defects associated with myotonic dystrophy

Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. We identified the small molecules pentamidine and neomycin B as compounds that disrupt MBNL1 binding to CUG repeats in vitro. We show in cell culture that pentamidine was able to reverse the missplicing of 2 pre-mRNAs affected in DM, whereas neomycin B had no effect. Pentamidine also significantly reduced the formation of ribonuclear foci in tissue culture cells, releasing MBNL1 from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of 2 pre-mRNAs in mice expressing expanded CUG repeats.

Role of RNA structure in regulating pre-mRNA splicing

Pre-mRNA splicing involves removing non-coding introns from RNA transcripts. It is carried out by the spliceosome, along with other auxiliary factors. In general, research in splicing has focused on the sequences within the pre-mRNA, without considering the structures that these sequences might form. We propose that the role of RNA structure deserves more consideration when thinking about splicing mechanisms. RNA structures can inhibit or aid binding of spliceosomal components to the pre-mRNA, or can increase splicing efficiency by bringing important sequences into close proximity. Recent reports have identified proteins and small molecules that can regulate splicing by modulating RNA structures, thereby expanding our knowledge of the mechanisms used to regulate splicing.

The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing

Myotonic dystrophy type 1 (DM1) is a genetic disorder linked to a (CTG)n repeat expansion in the 3′ untranslated region of the DMPK gene. Upon transcription in the nucleus, the CUG repeats form a stable RNA stem-loop that sequesters the RNA-binding protein MBNL1 from its normal function in the cell. MBNL1 regulates the alternative splicing of many pre-mRNAs, and upon MBNL1s sequestration, the alternative splicing of many genes is mis-regulated, leading to disease symptoms. MBNL1 is known to bind directly to at least 3 of the pre-mRNAs that it regulates, but how MBNL1 binding mechanistically regulates alternative splicing is unclear. Here, we demonstrate that MBNL1 controls the splicing of exon 5 in the cardiac troponin T (cTNT) pre-mRNA by competing directly with the essential splicing factor U2AF65 for binding at the 3′ end of intron 4. When U2AF65 is prevented from binding to the pre-mRNA, the U2 snRNP can no longer be recruited and the following exon is skipped. Furthermore, MBNL1 and U2AF65 appear to compete by binding to mutually exclusive RNA structures. When bound by splicing factors, the 3′ end of intron 4 can form either a stem-loop or a single-stranded structure. MBNL1 binds a portion of the intron as a stem-loop, whereas U2AF65 binds the same region in a single-strand structure. Mutations that strengthen the stem-loop decrease U2AF65 binding affinity and also repress exon 5 inclusion, independently of MBNL1. Thus, U2AF65 binding can be blocked either by MBNL1 binding or by the stabilization of RNA secondary structure.

MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs.

Superoxide Dismutase from the Eukaryotic Thermophile Alvinella pompejana: Structures, Stability, Mechanism, and Insights into Amyotrophic Lateral Sclerosis.

Prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. Alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 degrees C, with spikes up to 84 degrees C. Here, we used Cu,Zn superoxide dismutase (SOD) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability by supplying better stable mammalian homologs for structural biology and other biophysical characterizations than those from prokaryotic thermophiles. Identification, cloning, characterization, X-ray scattering (small-angle X-ray scattering, SAXS), and crystal structure determinations show that A. pompejana SOD (ApSOD) is superstable, homologous, and informative. SAXS solution analyses identify the human-like ApSOD dimer. The crystal structure shows the active site at 0.99 A resolution plus anchoring interaction motifs in loops and termini accounting for enhanced stability of ApSOD versus human SOD. Such stabilizing features may reduce movements that promote inappropriate intermolecular interactions, such as amyloid-like filaments found in SOD mutants causing the neurodegenerative disease familial amyotrophic lateral sclerosis or Lou Gehrigs disease. ApSOD further provides the structure of a long-sought SOD product complex at 1.35 A resolution, suggesting a unified inner-sphere mechanism for catalysis involving metal ion movement. Notably, this proposed mechanism resolves apparent paradoxes regarding electron transfer. These results extend knowledge of SOD stability and catalysis and suggest that the eukaryote A. pompejana provides macromolecules highly similar to those from humans, but with enhanced stability more suitable for scientific and medical applications.

Reducing levels of toxic RNA with small molecules.

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded noncoding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogues of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model.

The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements.

BackgroundMuscleblind-like 1 (MBNL1) is an alternative splicing factor containing four CCCH Zinc fingers (ZnFs). The sequestration of MBNL1 by expanded CUG and CCUG repeats is a major component in causing myotonic dystrophy. In addition to binding the structured expanded CUG and CCUG repeats; previous results suggested that MBNL1 binds single-stranded RNAs containing GC dinucleotides.ResultsWe performed a systematic analysis of MBNL1 binding to single-stranded RNAs. These studies revealed that a single GC dinucleotide in poly-uridine is sufficient for MBNL1 binding and that a second GC dinucleotide confers higher affinity MBNL1 binding. However additional GC dinucleotides do not enhance RNA binding. We also showed that the RNA sequences adjacent to the GC dinucleotides play an important role in MBNL1 binding with the following preference: uridines >cytidines >adenosines >guanosines. For high affinity binding by MBNL1, the distance between the two GC dinucleotides can vary from 1 to 17 nucleotides.ConclusionsThese results suggest that MBNL1 is highly flexible and able to adopt different conformations to recognize RNAs with varying sequence configurations. Although MBNL1 contains four ZnFs, only two ZnF - GC dinucleotide interactions are necessary for high affinity binding.

An Extended RNA Binding Site for the Yeast Branch Point-binding Protein and the Role of Its Zinc Knuckle Domains in RNA Binding

The highly conserved branch point sequence (BPS) of UACUAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.

Actinomycin D Specifically Reduces Expanded CUG Repeat RNA in Myotonic Dystrophy Models.

Myotonic dystrophy type 1 (DM1) is an inherited disease characterized by the inability to relax contracted muscles. Affected individuals carry large CTG expansions that are toxic when transcribed. One possible treatment approach is to reduce or eliminate transcription of CTG repeats. Actinomycin D (ActD) is a potent transcription inhibitor and FDA-approved chemotherapeutic that binds GC-rich DNA with high affinity. Here, we report that ActD decreased CUG transcript levels in a dose-dependent manner in DM1 cell and mouse models at significantly lower concentrations (nanomolar) compared to its use as a general transcription inhibitor or chemotherapeutic. ActD also significantly reversed DM1-associated splicing defects in a DM1 mouse model, and did so within the currently approved human treatment range. RNA-seq analyses showed that low concentrations of ActD did not globally inhibit transcription in a DM1 mouse model. These results indicate that transcription inhibition of CTG expansions is a promising treatment approach for DM1.