John R Buchan
Assistant Professor, BIO5 Institute
Assistant Professor, Molecular and Cellular Biology
Primary Department
Department Affiliations
(520) 626-1881
Research Interest
The control of gene expression is critical to nearly all aspects of cellular biology, from maintaining basic cell function and identity, to the ability of cells to respond to numerous signals that arise during processes such as development, exposure to pathogens or changes in the cellular environment. A key means by which all eukaryotic cells enact appropriate gene expression responses is to alter the function of messenger RNAs (mRNAs). mRNAs exist in different functional states, dependent upon the proteins bound to them. These states include translation (protein synthesis), repression (off state) and decay. The localization of an mRNAs can also affect its function, thus cells are offered an array of spatial and temporal mechanisms for gene expression control at the mRNA level.mRNAs can also cycle between these different functional states. For example, mRNAs exiting translation often accumulate in distinct mRNA-protein (mRNP) assemblies known as P-bodies and stress granules, from which they may ultimately return to translation again. P-bodies and stress granules are conserved throughout eukaryotes, contain important protein regulators of decay and translation, and thus affect gene expression control. In addition, they strongly resemble other important mRNP granules that function in embryogenesis (maternal granules) and memory formation (neuronal transport granules). Finally, these granules have numerous connections to disease, such as an involvement in RNA viral replication, elevated levels in certain cancer types, as well as the formation of apparently aberrant stress granules in neurodegenerative diseases.In the Buchan lab, we use yeast and cell line models to study the architecture of the cytoplasmic mRNP cycle. Our overall goal is to better understand what factors and processes affect the function of specific mRNAs, and how defects in this cycle may contribute to various disease states.

Publications

Zarnescu, D. C., Buchan, J. R., Liu, G., Coyne, A., Vaughan, S., & Pei, F. (2016). Endocytosis is a key regulator of TDP-43 toxicity and turnover. Nature Communications.

Amyotrophic lateral sclerosis (ALS) is a fatal motor-neuron degenerative disease. ALS-affected neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity surprisingly depend upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotordysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment or enhancement of endocytosis rates respectively. Thus, endocytosis dysfunction may be both an underlying cause of ALS pathology and a new therapeutic target.

Eshleman, N., Liu, G., McGrath, K., Parker, R., & Buchan, J. R. (2016). Defects in THO/TREX-2 function cause accumulation of novel cytoplasmic mRNP granules that can be cleared by autophagy. RNA (New York, N.Y.), 22(8), 1200-14.

The nuclear THO and TREX-2 complexes are implicated in several steps of nuclear mRNP biogenesis, including transcription, 3' end processing and export. In a recent genomic microscopy screen in Saccharomyces cerevisiae for mutants with constitutive stress granules, we identified that absence of THO and TREX-2 complex subunits leads to the accumulation of Pab1-GFP in cytoplasmic foci. We now show that these THO/TREX-2 mutant induced foci ("TT foci") are not stress granules but instead are a mRNP granule containing poly(A)(+) mRNA, some mRNP components also found in stress granules, as well several proteins involved in mRNA 3' end processing and export not normally seen in stress granules. In addition, TT foci are resistant to cycloheximide-induced disassembly, suggesting the presence of mRNPs impaired for entry into translation. THO mutants also exhibit defects in normal stress granule assembly. Finally, our data also suggest that TT foci are targeted by autophagy. These observations argue that defects in nuclear THO and TREX-2 complexes can affect cytoplasmic mRNP function by producing aberrant mRNPs that are exported to cytosol, where they accumulate in TT foci and ultimately can be cleared by autophagy. This identifies a novel mechanism of quality control for aberrant mRNPs assembled in the nucleus.

Liu, G., Lanham, C., Buchan, J. R., & Kaplan, M. E. (2017). High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots. PloS one, 12(3), e0174128.

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Liu, G., Coyne, A. N., Pei, F., Vaughan, S., Chaung, M., Zarnescu, D. C., & Buchan, J. R. (2017). Endocytosis regulates TDP-43 toxicity and turnover. Nature communications, 8(1), 2092.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology.

Liu, G., Pei, F., Yang, F., Li, L., Amin, A. D., Liu, S., Buchan, J. R., & Cho, W. C. (2017). Role of Autophagy and Apoptosis in Non-Small-Cell Lung Cancer. International journal of molecular sciences, 18(2).

Non-small-cell lung cancer (NSCLC) constitutes 85% of all lung cancers, and is the leading cause of cancer-related death worldwide. The poor prognosis and resistance to both radiation and chemotherapy warrant further investigation into the molecular mechanisms of NSCLC and the development of new, more efficacious therapeutics. The processes of autophagy and apoptosis, which induce degradation of proteins and organelles or cell death upon cellular stress, are crucial in the pathophysiology of NSCLC. The close interplay between autophagy and apoptosis through shared signaling pathways complicates our understanding of how NSCLC pathophysiology is regulated. The apoptotic effect of autophagy is controversial as both inhibitory and stimulatory effects have been reported in NSCLC. In addition, crosstalk of proteins regulating both autophagy and apoptosis exists. Here, we review the recent advances of the relationship between autophagy and apoptosis in NSCLC, aiming to provide few insights into the discovery of novel pathogenic factors and the development of new cancer therapeutics.