Regulation of Translation by NF1
Abstract
Neurofibromatosis Type I is cancer predisposition syndrome characterized by neurofibromas, benign tumors derived from peripheral nerve Schwann cells. Some of these tumors will progress to malignancy, which is a life-threatening outcome. Patients are also prone to optic gliomas and are predisposed to a range of other tumors. The gene that is mutated to cause Neurofibromatosis Type I, NF1, encodes a 319 kDa protein called neurofibromin (NF1). NF1 negatively regulates members of the Ras superfamily, oncogenes that are critical to the development of cancer. However, NF1 is a very large protein that also interacts with a number of other proteins and was recently found to directly regulate gene expression independent of Ras activity, so it clearly has a number of other, unknown functions that, when lost, result in tumor formation. To gain insight into new NF1 functions, we performed experiments to identify the proteins with which it interacts. This experiment identified at least 25 proteins that are integral components of ribosomes. Ribosomes are the organelles that translate mRNA into proteins, the last step in gene expression. We used an alternate method called proximity biotinylation also identified many of the same ribosomal proteins, supporting the original data. We were able to confirm that NF1 associates with ribosomes using another method that detected NF1 in a purified preparation of ribosomes. When we destroyed the ribosomes using the enzymes RNase A, this association was lost, confirming that NF1 associates with ribosomes. Neurofibromin is a regulatory protein, and loss of this regulatory function leads to changes in gene expression that ultimately result in Neurofibromatosis type 1. The observation that NF1 associates with ribosomes led us to the hypothesis that NF1 may regulate gene expression at the level of translation. If true, this would represent a completely novel mechanism of action for NF1. The question is how to test this hypothesis. We know that genes regulated at the level of translation have a high ratio of mRNA to protein. We reasoned that, if a gene were regulated this way by NF1, then this ratio would change upon loss of NF1. We therefore decided to measure the levels of both mRNA and proteins for all genes in the presence and absence of NF1 using the powerful and sensitive methods RNAseq, TRAPseq, and SILAC mass spectrum analysis. These experiments will identify changes in the mRNA and protein ratio that correlates with NF1 levels, thereby identifying genes that are regulated by NF1 at the level of translation. The discovery of NF1-mediated translational control would represent a major breakthrough in understanding a novel mechanism of action for NF1 and provide unique insight into the pathology of Neurofibromatosis Type I. If the hypothesis proves false, then we are still likely to identify novel genes regulated NF1 in human Schwann cells. This will also identify new biomarkers and possible drug targets for Neurofibromatosis Type 1. The results from these experiments will be a critical tool in understanding the function of NF1 and the consequences of NF1 mutation in the development of Neurofibromatosis Type I.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210196
Entities
People
- Robert F Hennigan
Organizations
- United States Army