Nuanced interactions in RNA production
A sensitive new method of extracting key molecules in our cells tells us more about the subtle interactions affecting protein synthesis, CSC research reveals.
Processes at the heart of the living cell, the biochemical mechanisms that keep us alive, must often be inferred from fragments of evidence. Scientific experiments that have gone before; banks of data stretching back decades; mechanisms postulated from chemical, physical and biological ‘common sense’, all contribute to our understanding of what’s happening inside us. Isolating a cell’s contents at specific stages of development is one of biology’s most successful approaches to unpicking the interactions of genes and proteins.
Francis Crick, co-discoverer of the DNA double helix, famously framed the cellular factory as a one-way flow: DNA makes RNA makes protein. But the cellular machines that make RNA are themselves proteins, in the form of enzymes. These in turn rely on other proteins to function properly. So rather than being a one-way street, a complex network of interactions must function together to keep us alive.
RNA polymerase II (RNAPII) is the enzyme that reads DNA sequences, copying these into strands of mRNA [messenger RNA], which tell the ribosome [protein factory] which amino acids to string together. Scientists need to isolate and purify enzymes like this to understand their workings. Yet, while extraction methods have efficiently isolated RNAPII, the enzyme’s behaviour in vitro will not precisely replicate that within its native environment of the cell. Researchers working in the CSC Genome Function group have circumvented the issue with a technique that preserves subtle interactions, giving us more information about how proteins interact with RNAPII and affect its function. Their work has been published online in the journal Molecular & Cellular Proteomics.
Ana Pombo, the study’s lead author, explains. “RNAPII dissociates from DNA when chromosomes are shutdown for cell division (mitosis). We speculated that certain proteins would continue to interact with RNAPII during this stage of the cell cycle.” Her team used mass spectrometry and fluorescence microscopy to identify the enzyme-protein interactions. “Our isolation method combined with immunoprecipitation led us to identify more than 400 proteins that associate with RNAPII. Previous research suggested there were at most 100 proteins.”
Among the newly identified proteins comprising the interaction network – dubbed the RNAPII interactome – the researchers found several with implications for disease. One of these, ALMS1, is mutated in Alström syndrome, a disorder that leads to obesity and diabetes. “We’ve shown unequivocally that ALMS1 interacts with RNAPII in cells and affects its activity,” says Ana. “This will open up avenues to further explore the molecular basis of metabolic disorders like Alström syndrome. Our datasets also provide a novel resource that will be useful for identifying additional roles for RNAPII in health and disease.”
SJ
Reference:
Moeller, A., Xie, S. Q., Hosp, F., Lang, B., Phatnani, H. P., James, S., Ramirez, F., Collin, G. B., Naggert, J. K., Babu, M. M., Greenleaf, A. L., Selbach, M., Pombo, A. (2011). Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease. Molecular & cellular proteomics, in press. Link to article.
