George W. Owttrim

RNA helicases and abiotic stress

RNA helicases function as molecular motors that rearrange RNA secondary structure, potentially performing roles in any cellular process involving RNA metabolism. Although RNA helicase association with a range of cellular functions is well documented, their importance in response to abiotic stress is only beginning to emerge. This review summarizes the available data on the expression, biochemistry and physiological function(s) of RNA helicases regulated by abiotic stress. Examples originate primarily from non-mammalian organisms while instances from mammalian sources are restricted to post-translational regulation of helicase biochemical activity. Common emerging themes include the requirement of a cold-induced helicase in non-homeothermic organisms, association and regulation of helicase activity by stress-induced phosphorylation cascades, altered nuclear–cytoplasmic shuttling in eukaryotes, association with the transcriptional apparatus and the diversity of biochemical activities catalyzed by a subgroup of stress-induced helicases. The data are placed in the context of a mechanism for RNA helicase involvement in cellular response to abiotic stress. It is proposed that stress-regulated helicases can catalyze a nonlinear, reversible sequence of RNA secondary structure rearrangements which function in RNA maturation or RNA proofreading, providing a mechanism by which helicase activity alters the activation state of target RNAs through regulation of the reaction equilibrium.

Regulation of Cold Shock-Induced RNA Helicase Gene Expression in the Cyanobacterium Anabaena sp. Strain PCC 7120

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30 degrees C but accumulate at 20 degrees 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 degrees 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.

Plant RNA helicases: linking aberrant and silencing RNA

RNA helicases are ATPases that are capable of rearranging RNA and ribonucleoprotein (RNP) structure, and they can potentially function in any aspect of RNA metabolism. The RNA helicase gene family of plant genomes is larger and more diverse than genome families observed in other systems and provides an ideal model for investigation of the physiological importance of RNA secondary structure rearrangement in plant development. Numerous plant RNA helicases are associated with a variety of physiological functions, but this review will focus on the thirteen RNA helicases associated with the metabolism of aberrant and silencing RNAs. The results emphasize the crucial role RNA helicase activity has in the regulation of mRNA quality control and gene expression in plant development.

CHARACTERIZATION OF THE TOBACCO EIF-4A GENE FAMILY

Characterization of cDNAs encoding eukaryotic translation initiation factor 4A (eIF-4A) indicates the expression of a minimum of ten related genes in tobacco leaf cells. The ten groups fall into two gene families, NeIF-4A2 and NeIF-4A3. The majority of the cDNAs exhibit significant sequence similarity to the NeIF-4A2 family at both the DNA and deduced amino acid levels. Northern analysis using specific probes indicates variable expression of four family members in various tobacco organs. Western analysis, using an anti-tobacco eIF-4A polyclonal antibody, reveals a complex pattern of immunologically related polypeptides of approximately 46 kDa. Subcellular fractionation suggests that at least one eIF-4A-related polypeptide is located in the chloroplast where it is ribosome-associated.

A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR

Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.

Autoregulation of RNA Helicase Expression in Response to Temperature Stress in Synechocystis sp. PCC 6803

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.

Inactivation of a Low Temperature-Induced RNA Helicase in Synechocystis sp. PCC 6803: Physiological and Morphological Consequences

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

A Synechocystis LexA-orthologue binds direct repeats in target genes

Although evidence for LexA‐orthologues, which do not regulate DNA damage repair, is accumulating, identification of binding sites and regulon members remains poorly characterized. In the cyanobacterium, Synechocystis sp. strain PCC 6803, we have recently identified a LexA‐related protein that regulates expression of the crhR RNA helicase gene. Here we show that the Synechocystis LexA‐orthologue binds as a dimer to 12 bp direct repeats containing a CTA‐N9‐CTA sequence conserved in two target genes, lexA and crhR. Characterization of this site provides the basis for identification of additional LexA targets and further evidence for LexAs divergence during evolution.

Highly conserved genes coding for eukaryotic translation initiation factor eIF-4A of tobacco have specific alterations in functional motifs

Eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase that is required for the binding of mRNA to ribosomes. Plant eIF-4A-like proteins are highly homologous to eIF-4As from yeast, mouse and Drosophila melanogaster. The pattern of intron-exon boundaries in eIF-4A-like genes are conserved within tobacco, but are not conserved with other organisms. Fixed spacings between the functionally important sequence motifs, GKT-PTRELA (72 bp), DEAD-SAT (81 bp) and SAT-HRIGR (426 bp), are conserved between plants, mouse, Drosophila and yeast.

RNA Helicases in Cyanobacteria: Biochemical and Molecular Approaches

RNA helicases are associated with every aspect of RNA metabolism and function. A diverse range of RNA helicases are encoded by essentially every organism. While RNA helicases alter gene expression, RNA helicase expression is itself regulated, frequently in response to abiotic stress. Photosynthetic cyanobacteria present a unique model system to investigate RNA helicase expression and function. This chapter describes methodology to study the expression and cellular localization of RNA helicases, providing insights into the metabolic pathway(s) in which these enzymes function in cyanobacteria. The approaches are applicable to other systems as well.