A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli, including extracellular osmolarity, the presence of procaine and interaction with an accessory protein, MzrA. Two major outer membrane porins, OmpF and OmpC, act as passive diffusion-limited pores that allow compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though the mechanisms by which EnvZ detects and processes the presence of various stimuli are a fundamental component of microbial physiology, they are not yet fully understood. Here, we assess the role of TM1 during signal transduction in response to the presence of extracellular osmolarity. Various mechanisms of transmembrane communication have been proposed including rotation of individual helices within the transmembrane domain, dynamic movement of the membrane-distal portion of the cytoplasmic domain and regulated intra-protein unfolding. To assess these possibilities, we have created a library of single-Cys-containing EnvZ proteins in order to facilitate sulfhydryl-reactivity experimentation. Our results demonstrate that the major TM1-TM1' interface falls along a single surface consisting of residue positions 19, 23, 26, 30 and 34. In addition, we show that Cys substitutions within the N- and C-terminal regions of TM1 result in drastic changes to EnvZ signal output. Finally, we demonstrate that core residues within TM1 are responsible for both TM1 dimerisation and maintenance of steady-state signal output. Overall, our results suggest that no major rearrangement of the TM1-TM1' interface occurs during transmembrane communication in response to extracellular osmolarity. We conclude by discussing these results within the frameworks of several proposed models for transmembrane communication.

61 62In Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent 63 groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in 64 clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression in 65Escherichia coli is EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes 66 periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the 67 ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane 68 (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/206888 doi: bioRxiv preprint first posted online EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-92 standing question of how transmembrane communication is performed by bacterial receptors. Our work 93 concludes with a framework that correlates receptor domain composition and signal transduction 94 mechanisms that could be employed by other research groups on their particular receptors of interest. 95 96 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Overexpression of G-patch proteins affects precursor ribosomal RNA processing 4. DISCUSSION 4.1. Characterisation of the orphan G-patch protein Cmg1 4.2. Prp43 at mitochondria in apoptosis 4.3. Recruitment of helicases to their target sites 4.4. Regulation of RNA helicases by cofactor competition 4.5. Conclusions