Baylor College of Medicine professor Thomas Cooper expressed his research lab’s interest in neuromuscular disease in the fifth lecture of the Natural Science and Mathematics Colloquium Series this semester, held on March 10.
In front of a large audience of students, professors, and community members in Schaefer Hall, Thomas Cooper, Professor of Pathology at Baylor, discussed developmental mechanisms that can lead to the symptoms of neuromuscular disease, including the more common myotonic dystrophy, in his presentation Developmentally-Regulated Alternative Splicing and Its Disruption in Neuromuscular Disease.
Cooper was not afraid to tell his audience how little is known about the mechanism of alternative splicing and myotonic dystrophy itself; rather, it serves as a major stimulus for his research.
“It’s exciting to find out how little we know,” he said.
Beginning with what is known about the genetics behind alternative splicing, Cooper discussed the central dogma of gene expression. DNA, a double-stranded sequence of nucleotides housed in the nucleus of a cell, undergoes a process known as transcription, where a single-stranded version of DNA (called mRNA) is created. This single-stranded sequenced undergoes processing in the nucleus before exiting into the cell’s cytoplasm as mature mRNA, which in turn undergoes a process known as translation to generate a functional protein.
While this sequence of events is generally followed, Cooper’s work focuses on the processing of pre-mRNA in the nucleus of the cell. RNA processing itself plays a major role in the expression of genes as proteins, simply due to the structure of the RNA itself. RNA is composed of, essentially, two types of gene sequences: introns, which do not code for a specified expressed gene, and exons, which do code for the expressed gene.
During pre-mRNA processing, the exon regions are precisely spliced, or cut, so that exons combine to make an exact coding sequence.
If splicing is not exact, and extra sequences appear in between the exons or if the exons are cut short, the resulting protein will usually be deformed in some way, as each building block of the final protein product (called an amino acid) is added based on sets of three nucleotides.
“If it misses a nucleotide or adds a nucleotide by mistake, it puts the sequence out of frame, and it’s not going to make a protein,” said Cooper.
Alternative splicing refers to the removal of certain introns and exons in various ways to make a different mRNA sequence, which in turn leads to a different protein. Given the extremely high number of possibilities of mRNA products, alternative splicing is regulated.
“A lot of proteome diversity changes not because of transcription,” said Cooper, “but what happens after the gene is transcribed.”
Cooper illustrated this point by describing the Dscam gene, a sequence found in chromosome 21 in humans that, with a certain mutation, can lead to Down Syndrome. The final mRNA product is composed of four main exons.
The first exon is chosen among 12 different exons, while the second is chosen among 48 different exons, the third among 33 different exons, and a fourth among two exons. Given the different combinations of these sequences, 38,000 possible mRNA sequences could be generated, up to 30,000 possible proteins due to alternative splicing alone.
This process can be regulated in multiple ways, including regulation of transcription. This would involve either increasing the activity (upregulating) or decreasing the activity (downregulating) of proteins involved in that process, or using positive and negative regulators to control which regions the spliceosomes (proteins and molecules that bind to splicing sites) bind.
Myotonic Dystrophy (DM) is the second most common form of muscular dystrophy, and is caused by a disruption in the alternative splicing mechanism. In this disease, the alternative splicing abnormality leads to the addition of nucleotide repeats.
Having more of these CTG repeats (named after the nucleotide bases that make up the mutation) causes a faster onset of the disease symptoms; while having 8-40 CTG repeats is normal, DM patients can have 80-2000 repeats.
Unlike other alternative splicing disorders, however, DM occurs in an intronic region, which does not even code for a portion of the final protein. Instead, the major problem occurs while the sequence is still in the pre-mRNA form.
The large repeat region is not able to leave the nucleus for further processing and translation, and instead serves as a toxic buildup in the nucleus. This buildup can be visualized using staining techniques, which show condensed matter (foci) in the nuclei of cells.
The CTG repeats, CUG repeats in pre-mRNA form, sequester (essentially, bind and deactivate) a splicing factor known as MBNL-1 (Muscleblind-like), which causes another splicing factor (CUGBP-1) to be induced (leading to the CUG sequence repeats and DM symptoms). CUGBP buildup can also be visualized, and correlates with MBNL buildup at the foci sites in the nuclei.
These abnormalities can lead to many issues, including problems in alternative splicing of other genes, faulty chloride channels in the muscle cells (which lead to the tensed muscles characteristic of DM), and insulin resistance (due to the inability to properly splice the adult form of the receptor).
Using a mouse model, Cooper’s lab studied the effects of altering the levels of CUGBP1 on the expression of DM-related symptoms. DM symptoms can be created in a mouse by injecting it with a chemical known as tamoxifen, which leads to the increased levels of CUGBP found in adult patients with DM.
By injecting the mice with BIS-IX, an inhibitor that slows production of CUGBP, the mice showed the same levels of mutated pre-mRNA, but not in a toxic buildup characteristic of DM; furthermore, the mortality of the mice was significantly reduced.
“PKC inhibitors [like BIS-IX] are being used as therapeutics for individuals with other diseases,” said Cooper.
Cooper concluded his presentation with a discussion of Baylor College itself, and the differences between being a researcher and a medical doctor. “The hardest part of research is all of the decisions you have to make,” he said. “You have to know how to make your best calls and know when to cut things off.”
“I thought Dr. Cooper did a good job at leading the audience into his area of expertise,” said Biochemistry professor Danielle Cass. “It seems that he tries to always keep in mind the patients with DM and how his research could benefit them.”
“I thought it was well-presented, but rather complicated,” said sophomore Steven Sheridan. “Considering the audience, I thought it was too advanced and fast-paced.”