SrmB is an RNA helicase from the DEAD-box family of proteins that has a known function assisting in the early steps of 50S ribosome subunit assembly. DEAD-box proteins are known to be involved in nearly every step of RNA metabolism;however, their specific mechanisms of action are largely unknown. The goal of the proposed project is to investigate the mechanism by which SrmB assists in 50S ribosome subunit assembly in vivo via quantitative mass spectrometry (QMS). First, we will use three QMS experiments (protein inventory, pulse labeling, and discovery proteomics) to characterize ribosome assembly intermediates of the ?SrmB E.coli strain, fractionally separated by sucrose gradient ultracentrifugation. These experiments will report on, respectively, the ribosomal protein composition of the intermediates compared to an intact ribosome internal standard, whether the intermediates are true on path-intermediates or degradation products, and their ribosome biogenesis cofactor composition. We will perturb the system by introducing plasmids expressing WT and mutant SrmB in the ?SrmB strain background and analyze the sucrose gradient fractions of these via the aforementioned QMS experiments. Second, we will introduce WT and mutant MBP-SrmB expression plasmids into ?SrmB cells and perform MBP tag pull downs with amylose resin of both crude lysate and sucrose gradient fractions in order to purify MBP-SrmB-associated ribosomal particles. Elution fractions will be analyzed by QMS, as described before, which will give a characterization of SrmB-associated ribosomal intermediates. Third, we will investigate the relationship between SrmB and other ribosomal proteins by perturbing ribosomal protein expression by overexpression in ?SrmB E. coli cells of ribosomal proteins S4 or S8, which are known autoregulators of polycistrionic operons for ribosomal proteins. Sucrose gradient fractions of these growths will be analyzed by QMS, and this analysis will allow us to determine functional connections between binding of specific ribosomal proteins and SrmB. All together, the experiments proposed in the three aims of this proposal will give a detailed picture of 50S subunit assembly from the standpoint of SrmB: that is, what ribosomal proteins and cofactors are required for SrmB to bind the 50S precursor particle, which require SrmB in order to engage with the 50S precursor particle, and which ribosomal proteins and cofactors bind and act upon the 50S precursor particle independently of SrmB. Both, misregulation of, the fundamental process of ribosome biogenesis and overexpression of DEAD-box proteins are observed in various disease states, including cancer. The studies proposed here will lead to a deeper and more detailed understanding of the molecular mechanism whereby RNA helicases operate on their targets as well as the complex process of ribosome biogenesis, which is paramount for the design of effective therapies for diseases in which they are implicated.
Ribosome assembly is one of the major metabolic activities in cells and is required for cell growth in all organisms;not surprisingly, substantial evidence points to the misassembly of ribosomes as a major cause of human disease, including cancer and other genetic diseases such as Diamond-blackfan anemia and the severe genetic disorder dyskeratosis congenita. DEAD-box proteins are overexpressed in various types of cancer cells and RNA helicases from related families are essential for the propagation of many viruses that cause human diseases. The project proposed here will help further knowledge of the mechanism of SrmB and DEAD-box helicases in general, as well as the process of ribosome assembly, which can be used to design effective drugs and therapies for diseases in which they are implicated.