John R Buchan
- Assistant Professor, Molecular and Cellular Biology
The over-arching goal of my research is to determine the functional relevance, assembly and clearance mechanisms of conserved mRNA-protein (mRNP) foci known as P-bodies (PBs) and stress granules (SGs). These cytoplasmic foci are conserved in all eukaryotes, and are thought to play key roles in regulating mRNA function. Recently, I demonstrated that SGs are targeted for clearance via a selective autophagic pathway. This is important as it may define a fundamental new mode by which cells control gene expression, and help explain the origin of diseases characterized by aberrant SG aggregates such as Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Dementia. In recent work, we are now pursuing the relevance of the endocytic pathway in clearance of TDP-43, a protein implicated in ALS pathology, thus the intersection of vesicular trafficking and RNA-protein biology is becoming a theme for us. These projects, combined with additional studies of how nuclear mRNA events (e.g. transcription, splicing, export) affect cytoplasmic mRNA function represent the main areas of study in my lab presently.
During my PhD and post-doc, I developed novel bioinformatic approaches to study the selective evolutionary pressures on transfer RNAs (tRNAs) and codon usage in prokaryotes, archae and eukaryotes, and how these could impact protein synthesis. I also discovered the existence of SGs in yeast, demonstrated that PBs can serve as nucleating sites for SG assembly, and obtained evidence of how cytoplasmic mRNAs transition through PBs, SGs and polysomes in an “mRNP cycle” that likely dictates mRNA function.
I currently supervise a lab that includes a post-doc, an assistant staff scientist, two graduate student, four undergraduates and a lab volunteer.
- Ph.D. Molecular Biology
- University of Aberdeen, UK, Aberdeen, United Kingdom
- Control of translation elongation
- B.S. Molecular and Cellular Biology
- University of Aberdeen, Aberdeen, United Kingdom
- Control of translation elongation
- Assistant Professor, University of Arizona, Tucson, Arizona (2014 - Ongoing)
- Research Associate, HHMI (2012 - 2013)
- Research Associate, HHMI (2006 - 2012)
Molecular and Cellular Biology, Genetics.
Gene expression, with a focus on mRNA biology. This includes translational control, mRNA decay, mRNA localization, and the role of mRNA-protein foci known as P-bodies and Stress granules. Also interested in vesicular trafficking pathways such as autopahgy and endocytosis, and how these may affect both stress granule clearance and ALS disease pathology.
Directed RsrchMCB 492 (Fall 2016)
DissertationMCB 920 (Fall 2016)
Honors ThesisBIOC 498H (Fall 2016)
Introduction to ResearchMCB 795A (Fall 2016)
Lab Presentations & DiscussionMCB 696A (Fall 2016)
CBIO GIDP Seminar SeriesCBIO 596H (Spring 2016)
Directed ResearchBIOC 492 (Spring 2016)
DissertationMCB 920 (Spring 2016)
Genetic & Molecular NetworksMCB 546 (Spring 2016)
Lab Presentations & DiscussionMCB 696A (Spring 2016)
Probl Solv/Genetic ToolsMCB 422 (Spring 2016)
ResearchMCB 900 (Spring 2016)
DissertationMCB 920 (Fall 2015)
Introduction to ResearchMCB 795A (Fall 2015)
Lab Presentations & DiscussionMCB 696A (Fall 2015)
ResearchMCB 900 (Fall 2015)
Directed RsrchMCB 392 (Spring 2015)
Genetic & Molecular NetworksMCB 546 (Spring 2015)
Honors ThesisBIOC 498H (Spring 2015)
Introduction to ResearchMCB 795A (Spring 2015)
Lab Presentations & DiscussionMCB 696A (Spring 2015)
ResearchMCB 900 (Spring 2015)
Senior CapstoneBIOC 498 (Spring 2015)
Directed RsrchMCB 392 (Fall 2014)
Introduction to ResearchMCB 795A (Fall 2014)
Lab Presentations & DiscussionMCB 696A (Fall 2014)
ResearchMCB 900 (Fall 2014)
Senior CapstoneBIOC 498 (Fall 2014)
- 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.More infoAmyotrophic 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., Lanham, C., Buchan, J. R., & Kaplan, M. E. (2017). High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots. PloS one, 12(3), e0174128.More infoSaccharomyces 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., 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).More infoNon-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.
- 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.More infoThe 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.
- Buchan, J. R., Kolaitis, R., Taylor, J. P., & Parker, R. (2013). Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell, 153(7), 1461-74.More infoStress granules and P bodies are conserved cytoplasmic aggregates of nontranslating messenger ribonucleoprotein complexes (mRNPs) implicated in the regulation of mRNA translation and decay and are related to RNP granules in embryos, neurons, and pathological inclusions in some degenerative diseases. Using baker's yeast, 125 genes were identified in a genetic screen that affected the dynamics of P bodies and/or stress granules. Analyses of such mutants, including CDC48 alleles, provide evidence that stress granules can be targeted to the vacuole by autophagy, in a process termed granulophagy. Moreover, stress granule clearance in mammalian cells is reduced by inhibition of autophagy or by depletion or pathogenic mutations in valosin-containing protein (VCP), the human ortholog of CDC48. Because mutations in VCP predispose humans to amyotrophic lateral sclerosis, frontotemporal lobar degeneration, inclusion body myopathy, and multisystem proteinopathy, this work suggests that autophagic clearance of stress granule related and pathogenic RNP granules that arise in degenerative diseases may be important in reducing their pathology.
- Buchan, J. R., Capaldi, A. P., & Parker, R. (2012). TOR-tured yeast find a new way to stand the heat. Molecular cell, 47(2), 155-7.More infoIn this issue, Takahara and Maeda (2012) discover that together, Pbp1 and sequestration of the TORC1 complex in cytoplasmic mRNP stress granules provides a negative regulatory mechanism for TORC1 signaling during stress.
- Buchan, J. R., Yoon, J., & Parker, R. (2011). Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae. Journal of cell science, 124(Pt 2), 228-39.More infoEukaryotic cells respond to cellular stresses by the inhibition of translation and the accumulation of mRNAs in cytoplasmic RNA-protein (ribonucleoprotein) granules termed stress granules and P-bodies. An unresolved issue is how different stresses affect formation of messenger RNP (mRNP) granules. In the present study, we examine how sodium azide (NaN(3)), which inhibits mitochondrial respiration, affects formation of mRNP granules as compared with glucose deprivation in budding yeast. We observed that NaN(3) treatment inhibits translation and triggers formation of P-bodies and stress granules. The composition of stress granules induced by NaN(3) differs from that of glucose-deprived cells by containing eukaryotic initiation factor (eIF)3, eIF4A/B, eIF5B and eIF1A proteins, and by lacking the heterogeneous nuclear RNP (hnRNP) protein Hrp1. Moreover, in contrast with glucose-deprived stress granules, NaN(3)-triggered stress granules show different assembly rules, form faster and independently from P-bodies and dock or merge with P-bodies over time. Strikingly, addition of NaN(3) and glucose deprivation in combination, regardless of the order, always results in stress granules of a glucose deprivation nature, suggesting that both granules share an mRNP remodeling pathway. These results indicate that stress granule assembly, kinetics and composition in yeast can vary in a stress-specific manner, which we suggest reflects different rate-limiting steps in a common mRNP remodeling pathway.
- Buchan, J. R., Nissan, T., & Parker, R. (2010). Analyzing P-bodies and stress granules in Saccharomyces cerevisiae. Methods in enzymology, 470, 619-40.More infoEukaryotic cells contain at least two types of cytoplasmic RNA-protein (RNP) granules that contain nontranslating mRNAs. One such RNP granule is a P-body, which contains translationally inactive mRNAs and proteins involved in mRNA degradation and translation repression. A second such RNP granule is a stress granule which also contains mRNAs, some RNA binding proteins and several translation initiation factors, suggesting these granules contain mRNAs stalled in translation initiation. In this chapter, we describe methods to analyze P-bodies and stress granules in Saccharomyces cerevisiae, including procedures to determine if a protein or mRNA can accumulate in either granule, if an environmental perturbation or mutation affects granule size and number, and granule quantification methods.
- Buchan, J. R., Muhlrad, D., & Parker, R. (2008). P bodies promote stress granule assembly in Saccharomyces cerevisiae. The Journal of cell biology, 183(3), 441-55.More infoRecent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.
- Buchan, J. R., & Parker, R. (2007). Molecular biology. The two faces of miRNA. Science (New York, N.Y.), 318(5858), 1877-8.
- Buchan, J. R., Aucott, L. S., & Stansfield, I. (2006). tRNA properties help shape codon pair preferences in open reading frames. Nucleic acids research, 34(3), 1015-27.More infoTranslation elongation is an accurate and rapid process, dependent upon efficient juxtaposition of tRNAs in the ribosomal A- and P-sites. Here, we sought evidence of A- and P-site tRNA interaction by examining bias in codon pair choice within open reading frames from a range of genomes. Three distinct and marked effects were revealed once codon and dipeptide biases had been subtracted. First, in the majority of genomes, codon pair preference is primarily determined by a tetranucleotide combination of the third nucleotide of the P-site codon, and all 3 nt of the A-site codon. Second, pairs of rare codons are generally under-used in eukaryotes, but over-used in prokaryotes. Third, the analysis revealed a highly significant effect of tRNA-mediated selection on codon pairing in unicellular eukaryotes, Bacillus subtilis, and the gamma proteobacteria. This was evident because in these organisms, synonymous codons decoded in the A-site by the same tRNA exhibit significantly similar P-site pairing preferences. Codon pair preference is thus influenced by the identity of A-site tRNAs, in combination with the P-site codon third nucleotide. Multivariate analysis identified conserved nucleotide positions within A-site tRNA sequences that modulate codon pair preferences. Structural features that regulate tRNA geometry within the ribosome may govern genomic codon pair patterns, driving enhanced translational fidelity and/or rate.
- Buchan, J. R. (2017, February). Endocytosis is a key regulator of TDP-43 toxicity and turnover. Phase Separation and RNA Processing as Drivers of Cancer and Neurodegenerative Disease. San Diego (La Jolla).
- Buchan, J. R. (2017, October). Endocytosis facilitates clearance of aggregation-prone proteins that underpin ALS pathology. Invited Seminar at UT-Health (McGovern Medical School), Houston. Houston: Microbiology and Molecular Genetics Dept., UT-Health (McGovern Medical School), Houston.
- Buchan, J. R. (2016, November). Surprising roles of autophagy and endocytosis in clearance of RNA-protein assemblies. Invited Seminar - UA Phoenix campus.More infoInvited seminar (by Kurt Gustin) at UA Phoenix campus -
- Buchan, J. R. (2015, February). P-bodies, Stress granules and their role in the mRNA life cycle. MCB/CBC/CMM joint seminar series. Kuiper Space Sciences, 308.More infoSeminar of lab research
- Buchan, J. R. (2014, October). The role of P-bodies and Stress granules in the complex life of mRNA. Joint Biology Research Retreat. Biosphere 2, Arizona: University of Arizona (MCB, CBC, CMM, and Immunology departments).
- Buchan, J. R. (2017, August). Endocytosis is a key regulator of TDP-43 aggregation, turnover and toxicity. Eukaryotic mRNA processing - CSHL. Cold Spring Harbor Laboratory, New York.
- Buchan, J. R. (2014, October). Dynamics, Regulation and Mechanism of Granulophagy. The Complex Life of mRNA. Heidelberg, Germany: EMBL.More infoPoster presentation at leading conference in the mRNA field.
- Buchan, J. R. (2014. mRNP granules: Assembly, function, and connections with disease.More infoMessenger ribonucleoprotein (mRNP) granules are dynamic, self-assembling structures that harbor non-translating mRNAs bound by various proteins that regulate mRNA translation, localization, and turnover. Their importance in gene expression regulation is far reaching, ranging from precise spatial-temporal control of mRNAs that drive developmental programs in oocytes and embryos, to similarly exquisite control of mRNAs in neurons that underpin synaptic plasticity, and thus, memory formation. Analysis of mRNP granules in their various contexts has revealed common themes of assembly, disassembly, and modes of mRNA regulation, yet new studies continue to reveal unexpected and important findings, such as links between aberrant mRNP granule assembly and neurodegenerative disease. Continued study of these enigmatic structures thus promises fascinating new insights into cellular function, and may also suggest novel therapeutic strategies in various disease states.
- Buchan, J. R., & Parker, R. (2009. Eukaryotic stress granules: the ins and outs of translation(pp 932-41).More infoThe stress response in eukaryotic cells often inhibits translation initiation and leads to the formation of cytoplasmic RNA-protein complexes referred to as stress granules. Stress granules contain nontranslating mRNAs, translation initiation components, and many additional proteins affecting mRNA function. Stress granules have been proposed to affect mRNA translation and stability and have been linked to apoptosis and nuclear processes. Stress granules also interact with P-bodies, another cytoplasmic RNP granule containing nontranslating mRNA, translation repressors, and some mRNA degradation machinery. Together, stress granules and P-bodies reveal a dynamic cycle of distinct biochemical and compartmentalized mRNPs in the cytosol, with implications for the control of mRNA function.
- Buchan, J. R., & Stansfield, I. (2007. Halting a cellular production line: responses to ribosomal pausing during translation(pp 475-87).More infoCellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.