The expression of defective RNAs can have pathological effects through ectopic or failed expression of proteins. Dr. James Anderson’s lab addresses fundamental questions about a mechanism required for the degradation of improperly formed RNAs. Nuclear RNA quality control and many normal RNA processing events in the eukaryotic nucleus critically depend on TRAMP complexes (an assembly of a non-canonical poly(A) polymerase, a Zn-knuckle protein, and a RNA helicase). There is an emerging picture suggesting that functions of the TRAMP complex, such as RNA recognition, adenylation and unwinding, are coordinated to ensure correct outcomes of processing and/or degradation of a wide variety of substrates. However, relatively little is understood regarding how the individual components of TRAMP interact to facilitate such coordination. Moreover, despite the key role of TRAMP in eukaryotic RNA metabolism, it remains unclear how TRAMP manages the remarkable feat of identifying and processing a specific, yet vastly diverse set of RNAs that are synthesized by different RNA polymerases, have no obvious common sequence or secondary structure, and share no common associated proteins. The goal of Dr. Anderson’s recently awarded NIH grant is to understand how TRAMP mediated degradation or processing of cellular RNAs impacts cellular health. To do this, they will use genetic screens to determine the function of a specific region of the RNA helicase portion of TRAMP complexes, develop methods to assay TRAMP substrate polyadenlation and structure within live yeast cells to work towards identifying features that make particular RNAs preferred substrates for the TRAMP complex, and explore potential roles of a Zn-knuckle protein in RNA binding and protein-protein interaction.
Dr. Anderson designed the study to provide extensive opportunities for both graduate student and undergraduate student participation. The common yeast, S. cerevisiae, has many well-known characteristics that make it a strong system for teaching and research, including its ease of culture and manipulation, short life cycle, ease of performing genetic analysis, and completely sequenced genome. Already, three undergraduate students, Justin Heilberger, Alexis Onderak and Joseph Burclaff had contributed to the project through their independent study projects. Yan Li is a graduate student in Dr. Anderson’s Lab, and has generated the preliminary data for screening of Mtr4p arch domain mutants. This work will be continued as her dissertation. Dr. Jane Dorweiler oversees the daily functions of the lab, and will be carrying out experiments to express, purify and characterize Air proteins, as well as purifying wild-type and hypomodified tRNAiMet from yeast for use in the in vitro studies.