Biology subjects

Hanson, M. R.

Publications and source records attributed to Hanson, M. R..

4 recordsLinked to original sources

bioRxiv2024

Accessory proteins increase the efficiency of RNA editing by Arabidopsis chloroplast editosomes

RNA editing modifies cytidines to uridines in plant organelle transcripts so that their sequences differ from the ones predicted from the genomic DNA. This process, conserved across most land plants, involves a family of RNA-binding proteins that has significantly expanded, the pentatricopeptide repeat (PPR)-containing proteins. In angiosperms, PPR proteins are found in editosomes associated with accessory proteins. The exact function of these accessory proteins has been unclear. Bacterial co-expression of an angiosperm synthetic factor and different accessory proteins, RIP2, RIP9, ORRM1 demonstrates their essential role in editing of an RNA target. The presence of ORRM1 and RIP2 or ORRM1 and RIP9 in bacteria with the PPR factor results in a target editing extent of 80%, which is similar to what is observed in planta. Accessory proteins increase the affinity of the PPR factor for the target RNA, likely the explanation of their role in improving editing efficiency. RNA-seq analysis of bacterial transcriptome in samples expressing various combinations of accessory proteins with the synthetic factor identified a total of 34 off-target editing events. Investigation of their upstream sequences that are recognized and bound by the synthetic factor allows the optimization of future designs to improve the specificity of this programmable RNA-editing factor.

molecular biology↗

bioRxiv2023

Dysregulation of extracellular vesicle protein cargo in female ME/CFS cases and sedentary controls in response to maximal exercise

In healthy individuals, physical exercise improves cardiovascular health and muscle strength, alleviates fatigue, and reduces risk of chronic diseases. Although exercise is suggested as a lifestyle intervention to manage various chronic illnesses, it negatively affects people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), who suffer from exercise intolerance. We hypothesized that altered extracellular vesicle (EV) signaling in ME/CFS patients after an exercise challenge may contribute to their prolonged and exacerbated negative response to exertion (post-exertional malaise). EVs were isolated by size exclusion chromatography from the plasma of 18 female ME/CFS patients and 17 age- and BMI-matched female sedentary controls at three time points: before, 15 minutes, and 24 hours after a maximal cardiopulmonary exercise test. EVs were characterized using nanoparticle tracking analysis and their protein cargo was quantified using Tandem Mass Tag-based (TMT) proteomics. The results show that exercise affects the EV proteome in ME/CFS patients differently than in healthy individuals and that changes in EV proteins after exercise are strongly correlated with symptom severity in ME/CFS. Differentially abundant proteins in ME/CFS patients vs. controls were involved in many pathways and systems, including coagulation processes, muscle contraction (both smooth and skeletal muscle), cytoskeletal proteins, the immune system, and brain signaling.

molecular biology↗

bioRxiv2022

Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation

ME/CFS is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we used single-cell RNA-seq (scRNA-seq) to examine immune cells in cohorts of patients and controls. Post-exertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. Thus, to detect changes coincident with PEM, we also performed scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients displayed dysregulation of classical monocytes suggestive of inappropriate differentiation and migration to tissue. We were able to identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlated with metrics of disease severity. Comparing the transcriptome at baseline and post-exercise challenge, we discovered patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation following exercise. HighlightsME/CFS is a debilitating disease with unknown causes. Here, we provide, for the first time, an extensive single cell resolution dataset detailing the gene expression programs of circulating immune cells of ME/CFS cases at baseline and after symptom provocation. We were able to detect robust dysregulation in certain immune cells from patients, with dysregulation of classical monocytes manifesting the strongest signal. Indeed, the fraction of aberrant monocytes in ME/CFS patients correlated with the degree of disease severity. Surprisingly, platelet transcriptomes were also altered in ME/CFS, and they were the only component of the immune system that showed large-scale changes following symptom provocation.

immunology↗

bioRxiv2019

Enzyme kinetics of tobacco Rubisco expressed in Escherichia coli varies depending on the small subunit composition

AbstractRubisco catalyzes the first step in carbon fixation and has been a strategic target to improve photosynthetic efficiency. In plants, Rubisco is a complex made up of eight large subunits encoded by a chloroplast gene, rbcL, and eight small subunits expressed from a nuclear gene family and targeted to chloroplast stroma. Biogenesis of Rubisco in plants requires a chaperonin system composed of Cpn60, Cpn60{beta} and Cpn20, which helps fold the large subunit, and multiple chaperones including RbcX, Raf1, Raf2 and BSD2, which help the dimerization of the folded large subunits and subsequent assembly with the small subunits into L8S8 holoenzymes. A recent study successfully assembled functional Arabidopsis Rubisco in Escherichia coli by co-expressing the two subunits with Arabidopsis chaperonins and chaperones (Aigner et al., 2017). In this study, we modified the expression vectors used in that study and adapted them to express tobacco Rubisco by replacing the Arabidopsis genes with tobacco ones. Next, we surveyed the small subunits present in tobacco, co-expressed each with the large subunit and successfully produced active tobacco enzymes composed of different small subunits in E. coli. These enzymes produced in E. coli have carboxylation kinetics very similar to that of the native tobacco Rubisco. We also produced tobacco Rubisco with a recently discovered trichome small subunit in E. coli and found that it has a higher catalytic rate and a lower CO2 affinity compared to the enzymes with other small subunits. Our improvements in the E. coli Rubisco expression system will allow us to probe features of both the chloroplast and nuclear-encoded subunits of Rubisco that affect its catalytic rate and CO2 specificity.

plant biology↗