The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter.

IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

Differential Centrifugation to Enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus.

Bacterial RNP bodies (BR bodies) contain the mRNA decay machinery, but the collection of associated RNAs and proteins are poorly defined. Here, we present a protocol for the rapid differential centrifugation-based enrichment of BR bodies from Caulobacter crescentus cells. As native BR bodies are highly labile and dissociate by degrading internal mRNAs, an active site mutant of RNase E, which blocks dissolution of BR bodies, allows BR-body stabilization during enrichment. For complete details on the use and execution of this protocol, please refer to Al-Husini et al. (2020).

Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes.

Peptides have important biomedical applications, but poor correlation between in vitro and in vivo activities can limit their development for clinical use. The ability to generate peptides and monitor their expression with new mass spectrometric methods and biological activities in vivo would be an advantage for the discovery and improvement of peptide-based drugs. In this study, a plasmid-based system was used to express the ribosome-targeting peptide oncocin (19 amino acids, VDKPPYLPRPRPPRRIYNR) and to determine its direct antibacterial effects on Escherichia coli. Previous biochemical and structure studies showed that oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results that are consistent with those of in vitro studies but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides reveals which amino acids are important for antimicrobial activity. Overall, the in vivo peptide expression system can be used to investigate biological activities and interactions of peptides with their targets within the cellular environment and to separate contributions of the sequence to cellular transport. This strategy has future applications for improving the effectiveness of existing peptides and developing new peptide-based drugs.

Phase-separated bacterial ribonucleoprotein bodies organize mRNA decay.

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BR-bodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Exposure to multiple career pathways by biomedical doctoral students at a public research university.

The Broadening Experiences in Scientific Experiences (BEST) program at Wayne State University was designed to increase doctoral students' awareness of multiple employment sectors beyond academia, improve their knowledge of transferable skills required to succeed in any career path, provide opportunities to explore diverse career paths, and gain in-depth knowledge about those paths using experiential learning opportunities. We devised a three-phase program that ranged from providing students with a broad introduction to multiple career opportunities to immersive experiential learning in a specific career sector. Importantly, program content was developed and delivered by alumni and industry experts in five employment sectors-business/industry, communication, government, law/regulatory affairs, and undergraduate/PUI teaching-in partnership with WSU faculty. This article provides data on two notable outcomes: doctoral students participate equally in BEST activities regardless of gender, race, and citizenship status, and student participation in BEST activities did not correlate with lower GRE ratings, lower GPA, or increased time-to-degree. Further, a "halo" effect of the program is evidenced by participation of students from all disciplines, not just the biomedical sciences. Centralizing BEST activities within the Graduate School will allow faculty and individual programs to save resources and time.

Visualization of gender, race, citizenship and academic performance in association with career outcomes of 15-year biomedical doctoral alumni at a public research university.

It has long been thought that biomedical doctoral students pursue careers primarily as tenure-track/tenured faculty at research institutions. Recent reports showed, however, that the majority of biomedical doctoral alumni engage in a variety of careers. Wayne State University (WSU) undertook a project to understand the career trajectories of its biomedical doctoral alumni to create programs to better prepare its students for careers in multiple pathways. Data were collected on career outcomes of WSU's biomedical doctoral alumni who graduated in a 15-year period from 1999-2014. Careers were classified into three tiers by Employment Sector, Career Types and Job Functions and career paths were examined by alumni gender, race, U.S. citizenship status, and association with certain academic characteristics. Several statistically significant differences in career paths among all demographics were found. For example, women were more likely to be in teaching and providing healthcare, men in faculty and research; Black alumni pursued careers in Government at higher rates and Whites in For-Profit careers; Asians and non-U.S. citizens spent more time in training positions than others. There was no association of academic characteristics such as GRE, GPA, and Time-to-Degree completion with careers in the two largest sectors of Academia or For-profit. Since our trainees are engaged in this rich variety of careers essential to advancing biomedical science and research nationally, it is imperative for the graduate training community to embrace all careers as successful, and transform the model for biomedical doctoral training to foster student success across this broad career spectrum.