SUMMARY Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Bacterial pathogens have evolved sophisticated signal transduction systems to coordinately control the expression of virulence determinants. For example, the human pathogen Vibrio cholerae is able to respond to host environmental signals by activating transcriptional regulatory cascades. The host signals that stimulate V. cholerae virulence gene expression, however, are still poorly understood. Previous proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression. In this study, we found that under oxygen-limiting conditions, an environment similar to the intestines, V. cholerae virulence genes are highly expressed. We show that anaerobiosis enhances dimerization and activity of AphB, a transcriptional activator that is required for the expression of the key virulence regulator TcpP, which leads to the activation of virulence factor production. We further show that one of the three cysteine residues in AphB, C 235 , is critical for oxygen responsiveness, as the AphB C235S mutant can activate virulence genes under aerobic conditions in vivo and can bind to tcpP promoters in the absence of reducing agents in vitro. Mass spectrometry analysis suggests that under aerobic conditions, AphB is modified at the C 235 residue. This modification is reversible between oxygen-rich aquatic environments and oxygen-limited human hosts, suggesting that V. cholerae may use a thiol-based switch mechanism to sense intestinal signals and activate virulence. thiol-modification | virulence activatorsT he Gram-negative bacterium Vibrio cholerae, the causative agent of the acute, dehydrating diarrheal disease cholera, has figured prominently in the history of infectious diseases as a cause of periodic, deadly pandemics. V. cholerae resides in aquatic environments between epidemics, and human infection normally starts with the ingestion of contaminated food or water. Vibrio cells surviving passage through the acidic gastric environment enter the small intestine, where they must produce an array of virulence factors including cholera toxin (CT) and the toxin co-regulated pilus (TCP) that are transcriptionally regulated by multiple systems (1). The primary, direct transcriptional activator of virulence genes is ToxT, whose transcription is regulated by the ToxRS and TcpPH proteins. Two additional activators encoded by unlinked genes, AphA and AphB, regulate the transcription of tcpPH.The environmental cues within the host and their effect on the expression of virulence genes in V. cholerae in vivo remain poorly characterized. It has been shown that anaerobiosis serves as one of the host environmental factors that modulate virulence factor production (2). This is not surprising because it is generally presumed that the oxygen concentration in the intestine is low (3). A recent report showed that under anaerobic conditions, tcpP expression is higher and this effect depends on AphB (4). However, whether and how this AphB-mediated tcpP expression contributes to anaerobic virulence indu...

Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.

SUMMARY The Arabidopsis thaliana root epidermis is comprised of two cell types, hair and nonhair cells, which differentiate from the same precursor. Although the transcriptional programs regulating these events are well studied, post-transcriptional factors functioning in this cell fate decision are mostly unknown. Here, we globally identify RNA-protein interactions and RNA secondary structure in hair and nonhair cell nuclei. This analysis reveals distinct structural and protein binding patterns across both transcriptomes, allowing identification of differential RNA binding protein (RBP) recognition sites. Using these sequences, we identify two RBPs that regulate hair cell development. Specifically, we find that SERRATE functions in a microRNA-dependent manner to inhibit hair cell fate, while also terminating growth of root hairs mostly independent of microRNA biogenesis. Additionally, we show that GLYCINE-RICH PROTEIN 8 promotes hair cell fate while alleviating phosphate starvation stress. In total, this global analysis reveals novel post-transcriptional regulators of plant root epidermal cell fate.

In this minireview, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the “forward” and “reverse” electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302 in R. capsulatus and Y278 in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra molecular organization of bacterial and mitochondrial respiratory chains (i. e., respirasomes) and their potential disease-associated protective roles.