RNA helicases are ubiquitous enzymes whose modification of RNA secondary structure is known to regulate RNA function. The pathways controlling RNA helicase expression, however, have not been well characterized. Expression of the cyanobacterial RNA helicase, crhR, is regulated in response to environmental signals that alter the redox poise of the electron transport chain, including light and temperature. Here we analyze crhR expression in response to alteration of abiotic conditions in wild type and a crhR mutant, providing evidence that CrhR autoregulates its own expression through a combination of transcriptional and post-transcriptional mechanisms. Temperature regulates crhR expression through alteration of both transcript and protein half-life which are significantly extended at low temperature (20°C). CrhR-dependent mechanisms regulate both the transient accumulation of crhR transcript at 20°C and stability of the CrhR protein at all temperatures. CrhR-independent mechanisms regulate temperature sensing and induction of crhR transcript accumulation at 20°C and the temperature regulation of crhR transcript stability, suggesting CrhR is not directly associated with crhR mRNA turnover. Many of the processes are CrhR- and temperature-dependent and occur in the absence of a correlation between crhR transcript and protein abundance. The data provide important insights into not only how RNA helicase gene expression is regulated but also the role that rearrangement of RNA secondary structure performs in the molecular response to temperature stress. We propose that the crhR-regulatory pathway exhibits characteristics similar to the heat shock response rather than a cold stress-specific mechanism.

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.The ability of organisms to rearrange nucleic acid secondary structure is crucial for cellular function and is catalyzed by a diverse range of proteins or protein complexes that facilitate nucleic acid annealing and unwinding. Two protein families, nucleic acid-binding proteins and helicases, catalyze these reactions. RNA-binding proteins are structurally unrelated to helicases (1, 2) and rearrange RNA secondary structure through chaperone-mediated annealing or unwinding in ATPindependent reactions (3, 4). Helicases have been classified into five major groups based on characteristic amino acid motifs with the two largest families, superfamilies 1 and 2, composed of RNA and DNA helicases (5). The other helicase families include proteins possessing fewer conserved motifs and having different substrate specificities.Biochemically, helicases function as ATP-driven molecular motors, catalyzing NTP-dependent nucleic acid duplex destabilization or strand displacement (6, 7). Although a number of RNA helicases possess RNA unwinding activity in vitro, only three have been reported to exhibit intrinsic RNA annealing activity, the highly related yeast nuclear DEAD-box RNA helicases, p68 and p72 (8), and the nucleolar DExD-box protein, RNA helicase II/Gu (9, 10). Although these helicases unwind dsRNA, 1 the RNA substrates on which they catalyze RNA annealing differ with p68/72 capable of annealing complementary ssRNA...

Dymowska AK, Schultz AG, Blair SD, Chamot D, Goss GG. Acidsensing ion channels are involved in epithelial Na ϩ uptake in the rainbow trout Oncorhynchus mykiss. Am J Physiol Cell Physiol 307: C255-C265, 2014. First published June 4, 2014 doi:10.1152/ajpcell.00398.2013.-A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na ϩ uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4=,6-diamidino-2-phenylindole and diminazene decreased Na ϩ uptake in adult rainbow trout in a dose-dependent manner, with IC 50 values of 0.12 and 0.96 M, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na ϩ -K ϩ -ATPase (␣5-subunit) antibody demonstrated that the trout ASIC localizes to Na ϩ /K ϩ -ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na ϩ uptake from dilute freshwater in the gill of rainbow trout. sodium uptake; gill; acid-sensing ion channels; mitochondrion-rich cells; fish; ionoregulation FISHES LIVING IN FRESHWATER (FW) must actively take up Na ϩ against a steep concentration gradient. Na ϩ uptake occurs via specialized mitochondrion-rich cells (MRCs), located on the fish gill epithelium (26, 29), and was initially proposed to be linked to NH 4 ϩ /H ϩ excretion (26). Subsequent studies have described two models of Na ϩ transport (for review see Refs. 8 and 18). In the first proposed model, Na ϩ is exchanged for H ϩ via an electroneutral Na ϩ /H ϩ exchanger (NHE) located on the apical membrane of MRCs in fish gill epithelia. The identification of multiple NHEs in the gills of zebrafish (Danio rerio) (51), Osorezan dace (Tribolodon hakonensis) (15), rainbow trout (Oncorhynchus mykiss) (7,19), and tilapia (Oreochromis mossambicus) (48) by immunocytochemistry, Western blot analysis, and RT-PCR, supports this model. However, significant thermodynamic constraints associated with the function of NHEs at very low ion concentrations (Na ϩ Ͻ0.1 mM) and low environmental pH (pH Ͻ5) (1, 34) suggest that fishes living in very soft and poorly buffered water would not be able to rely on a NHE-based mechanism for sufficient Na ϩ uptake. Recently, the NHE model was revised, whereby the ammonia transporter [rhesus (Rh) protein] present on the apical membrane of MRCs (30, 31) forms a functional metabolon with NHE2/3 (50). The revised model does alleviate the thermodynamic constraints associated with a low-pH environment, but not those imposed by low Na ϩ concentrations in the FW aquatic environment (6). Therefore, it is unlikely that this mechanism is the sole contributor to Na ϩ uptake by FW fishes living in low-ionic-strengt...

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30°C but accumulate at 20°C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20°C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.

Two Nicotiana plumbaginifolia cDNA clones, NeIF-5A1 and NeIF-5A2, encoding eukaryotic translation initiation factor eIF-5A (formerly called eIF-4D) were cloned by heterologous screening with Dictyostelium and human eIF-5A probes. eIF-5A is the only protein known to contain a unique amino acid modification, hypusine. Comparison of the Nicotiana deduced amino acid sequences with those of other eIF-5A polypeptides reveals conservation throughout the coding sequence, especially in the region of the hypusine residue. Transcript analysis reveals that NeIF-5A1 is preferentially expressed in photosynthetic tissues, while NeIF-5A2 is constitutively expressed in all plant tissues examined. A polyclonal antibody was raised against NeIF-5A1 overexpressed in E. coli. NeIF-5A1 antiserum crossreacts with an 18 kDa polypeptide doublet in all tobacco tissues examined. At least one polypeptide of ca. 18 kDa from a diversity of higher and lower plants crossreacts with NeIF-5A1 antiserum.