Biology subjects

Bryant, C. J.

Publications and source records attributed to Bryant, C. J..

4 recordsLinked to original sources

bioRxiv2022

Measurement of plant water status via static uniaxial compression of the leaf lamina

Turgor pressure is an essential, but difficult to measure indicator of plant water status. Turgor has been quantified by localised compression of cells or tissues, but a simple method to perform these measurements is lacking. We hypothesized that changes in leaf turgor pressure can be monitored by uniaxially compressing the leaf lamina and measuring the mechanical stress under a constrained thickness (stress relaxation); and that changes in leaf water content can be monitored by measuring the thickness of the leaf lamina compressed under a constant force (creep). Using a custom-built leaf squeeze-flow rheometer, we performed different compression tests on leaves from thirteen plant species. The equilibrium mechanical stress measured during stress relaxation was correlated with leaf turgor pressure (R2 > 0.95) and thus with leaf water potential (R2 > 0.94); the equilibrium leaf thickness measured during creep was correlated with relative water content (R2 > 0.74). The coefficients of these relationships were related to the leaf osmotic pressure at the turgor-loss point. An idealised average-cell model suggests that, under isothermal conditions, the bulk cell stiffness during compression is largely determined by the leaf osmotic pressure. Our study presents an inexpensive, accessible and automatable method to monitor plant water status non-invasively.

plant biology↗

bioRxiv2022

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for ribosome biogenesis, and as an interactor with the PeBoW complex (PES1, BOP1, WDR12) in high-throughput affinity purifications. The PeBoW complex has been shown to be required for pre-28S rRNA processing. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in mammalian cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We also demonstrate that RSL24D1 is physically complexed with RNA polymerase I, revealing a connection between large ribosomal subunit biogenesis and rDNA transcription. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large subunit biogenesis factors in pre-rRNA transcription control.

biochemistry↗

bioRxiv2021

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis, and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting ribosome biogenesis. However, there is a need for a more direct high-throughput assay for nucleolar function to further evaluate hits. Previous reports have monitored nucleolar RNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay for nucleolar function which enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay utilizes two siRNA controls, a negative non-targeting siRNA control (siNT) and a positive siRNA control targeting POLR1A (siPOLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dissociation. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of ribosome biogenesis in health and disease.

cell biology↗

bioRxiv2021

The human RBM10 gene dually encodes a repressor of ribosome biogenesis that downregulates cell proliferation

Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins.

molecular biology↗