Ross Buchan

Interests

Teaching

Molecular and Cellular Biology, Genetics.

Research

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.

Courses

Scholarly Contributions

Chapters

• Buchan, J. R. (2008). Nitric oxide in experimental autoimmune uveitis. In Free Radicals in Ophthalmic Disease(pp 107-119). Informa.
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  Nitric oxide in experimental autoimmune uveitis. J. Liversidge, S.Gordon, A. Dick, M. Robertson, and R. Buchan. Free Radicals in Ophthalmic Disease.Eds M. Zeirhut, E. Cadenas, N. Rao. Informa (New York) p107-­‐119(2008)

Journals/Publications

• Bearss, J. J., Padi, S. K., Singh, N., Cardo-Vila, M., Song, J. H., Mouneimne, G., Fernandes, N., Li, Y., Harter, M. R., Gard, J. M., Cress, A. E., Peti, W., Nelson, A. D., Buchan, J. R., Kraft, A. S., & Okumura, K. (2021). EDC3 phosphorylation regulates growth and invasion through controlling P-body formation and dynamics. EMBO reports, e50835.
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  Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.
• Fernandes, N., & Buchan, J. R. (2021). RNAs as Regulators of Cellular Matchmaking.. Frontiers in molecular biosciences, 8, 634146. doi:10.3389/fmolb.2021.634146
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  RNA molecules are increasingly being identified as facilitating or impeding the interaction of proteins and nucleic acids, serving as so-called scaffolds or decoys. Long non-coding RNAs have been commonly implicated in such roles, particularly in the regulation of nuclear processes including chromosome topology, regulation of chromatin state and gene transcription, and assembly of nuclear biomolecular condensates such as paraspeckles. Recently, an increased awareness of cytoplasmic RNA scaffolds and decoys has begun to emerge, including the identification of non-coding regions of mRNAs that can also function in a scaffold-like manner to regulate interactions of nascently translated proteins. Collectively, cytoplasmic RNA scaffolds and decoys are now implicated in processes such as mRNA translation, decay, protein localization, protein degradation and assembly of cytoplasmic biomolecular condensates such as P-bodies. Here, we review examples of RNA scaffolds and decoys in both the nucleus and cytoplasm, illustrating common themes, the suitability of RNA to such roles, and future challenges in identifying and better understanding RNA scaffolding and decoy functions.
• Klionsky, D. J., Abdel-Aziz, A. K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M. H., Abudu, Y. P., Acevedo-Arozena, A., Adamopoulos, I. E., Adeli, K., Adolph, T. E., Adornetto, A., Aflaki, E., Agam, G., Agarwal, A., Aggarwal, B. B., Agnello, M., , Agostinis, P., et al. (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy, 1-382.
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  In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
• Buchan, J. R., Buchanan, A., Pei, F., Byrd, A., Liu, G., Warner, A. N., & Basha, E. (2020). Cdc48/VCP and Endocytosis Regulate TDP-43 and FUS Toxicity and Turnover. Molecular and Cellular Biology, 40(4). doi:10.1128/mcb.00256-19
• Eshleman, N., Luo, X., Capaldi, A., & Buchan, J. R. (2020). Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast. RNA (New York, N.Y.), 26(1), 10-18.
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  Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA-protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls "Tet On/Tet Off" regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.
• Fernandes, N., & Buchan, J. R. (2020). RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly. Nucleic acids research, 48(11), 6265-6279.
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  P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.
• Fernandes, N., Nero, L., Lyons, S. M., Ivanov, P., Mittelmeier, T. M., Bolger, T. A., & Buchan, J. R. (2020). Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation. Biomolecules, 10(10).
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  Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Interestingly, in SG assembly mutant cells (), TDP-43 is enriched in nucleoli. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.
• Liu, G., Byrd, A., Warner, A. N., Pei, F., Basha, E., Buchanan, A., & Buchan, J. R. (2020). Cdc48/VCP and Endocytosis Regulate TDP-43 and FUS Toxicity and Turnover. Molecular and cellular biology, 40(4).
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  Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mislocalize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mislocalization and aggregation are implicated in ALS pathology, though the mechanism(s) of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids the turnover (i.e., protein degradation) of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 cofactor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in Cdc48 physically interacts and colocalizes with TDP-43, as does VCP, in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function but not on autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identify a role for Cdc48/VCP and endocytic function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting the cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.
• Fernandes, N., Eshleman, N., & Buchan, J. R. (2018). Stress Granules and ALS: A Case of Causation or Correlation?. Advances in neurobiology, 20, 173-212.
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  Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by cytoplasmic protein aggregates within motor neurons. These aggregates are linked to ALS pathogenesis. Recent evidence has suggested that stress granules may aid the formation of ALS protein aggregates. Here, we summarize current understanding of stress granules, focusing on assembly and clearance. We also assess the evidence linking alterations in stress granule formation and dynamics to ALS protein aggregates and disease pathology.
• 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.
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  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., Lanham, C., Buchan, J. R., & Kaplan, M. E. (2017). High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots. PloS one, 12(3), e0174128.
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  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., 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).
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  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.
• 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.
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  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.
• Buchan, J. R. (2014). mRNP granules. Assembly, function, and connections with disease. RNA biology, 11(8), 1019-30.
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  Messenger 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., Kolaitis, R., Taylor, J. P., & Parker, R. (2013). Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell, 153(7), 1461-74.
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  Stress 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.
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  In 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.
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  Eukaryotic 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.
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  Eukaryotic 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.
• Parker, R., & Buchan, J. R. (2009). Eukaryotic stress granules: the ins and outs of translation.. Molecular cell, 36(6), 932-41. doi:10.1016/j.molcel.2009.11.020
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  The 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., Muhlrad, D., & Parker, R. (2008). P bodies promote stress granule assembly in Saccharomyces cerevisiae. The Journal of cell biology, 183(3), 441-55.
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  Recent 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.
• Stansfield, I., & Buchan, J. R. (2007). Halting a cellular production line: responses to ribosomal pausing during translation.. Biology of the cell, 99(9), 475-87. doi:10.1042/bc20070037
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  Cellular 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.
• 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.
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  Translation 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.

Presentations

• Buchan, J. R. (2021, October). Novel mechanisms of TDP-43 proteostasis: Impacts for ALS. Biochemistry department journal club.
• Buchan, J. R. (2021, September). Stress granules and P-bodies - Roles in Cancer Biology (the extended cut). Cancer GIDP seminar. Kiewit Auditorium, UA.
• Buchan, J. R. (2021, September). Stress granules and P-bodies - Roles in Cancer Biology. Cancer Rounds seminar series. Kiewit Auditorium, U of Arizona: Cancer Center.
• Buchan, J. R. (2020, Nov 5th-6th). TDP-43 proteostasis revisited; role of endocytosis and stress granules. Arizona ALS Symposium 2020. Virtual.
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  Seminar on recent lab work
• Buchan, J. R. (2019, January). Novel P-body assembly mechanisms in yeast - a role for RPS28B 3'UTR scaffolding. "The RNA Revolution" - Cell and Molecular Genetics training grant symposium - UCSD/Salk Institute. UCSD Campus, San Diego.
• Buchan, J. R. (2019, October). A novel role for an mRNA 3'UTR in driving P-body assembly. Genetic GIDP seminar. Bio5, University of Arizona.
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  Seminar for Genetics GIDP membership
• Buchan, J. R. (2018, August). Endocytic function regulates TDP-43 toxicity, turnover and aggregation in multiple models. Living Like Lou - Emerging ALS Investigators symposium, University of Pittsburgh Brain Institute. University of Pittsburgh, PA: Living Like Lou Foundation.
• Buchan, J. R. (2018, December). Endocytic function regulates TDP-43 toxicity, turnover and aggregation in multiple models. Invited seminar at Hunter College, CUNY, New York. Hunter College campus, New York.
• Buchan, J. R. (2018, February). Endocytosis facilitates clearance of aggregation-prone proteins that underpin ALS pathology. 1st International Symposium of Amyotrophic Lateral Sclerosis - Alagoas in Motion. Maceio, Brazil.
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  Invited speaker - also served in role as international meeting organizer.
• Buchan, J. R. (2018, September). Endocytic function regulates TDP-43 toxicity, turnover and aggregation in multiple models. 3rd Annual Arizona ALS Symposium, Flagstaff. Flagstaff, Arizona.
• Buchan, J. R. (2017, Feb). 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.
• 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, December). Endocytosis is a key regulator of TDP-43 toxicity and turnover. Keystone conference: Cellular Stress Reponses and Infectious Agents. Santa Fe: Keystone.
• Buchan, J. R. (2016, November). Surprising roles of autophagy and endocytosis in clearance of RNA-protein assemblies. Invited Seminar - UA Phoenix campus.
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  Invited seminar (by Kurt Gustin) at UA Phoenix campus -
• Buchan, J. R. (2016, September). Endocytosis affects toxicity and turnover of TDP-43. Arizona ALS Symposium. Scottsdale: Barrow Neurological Institute.
• 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.
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  Seminar 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).

Poster Presentations

• 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.
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  Poster presentation at leading conference in the mRNA field.

Reviews

• Fernandes, N., & Buchan, J. R. (2021. RNAs as regulators of cellular matchmaking.
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  Nikita's thesis introduction was converted into a review that has just been accepted for publication in Frontiers in Molecular Biosciences. The review focuses on the numerous ways in which RNA molecules can function as scaffolds or decoys in a wide array of cellular process. The theme/interest is inspired by Nikita's work on mRNA 3'UTRs scaffolding PBs and nascent protein interactions.It's a comprehensive work. About 8000 words, 4 figures, 1 extensive table.The review is accepted, and we're just tidying up the format of reference list before an online version will be made available.
• Buchan, J. R. (2014. mRNP granules: Assembly, function, and connections with disease.
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  Messenger 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).
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  The 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).
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  Cellular 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.