Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus

ABSTRACT In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

Download Full-text

Both Enolase and the DEAD-Box RNA Helicase CrhB Can Form Complexes with RNase E in Anabaena sp. Strain PCC 7120

Applied and Environmental Microbiology ◽

10.1128/aem.00425-20 ◽

2020 ◽

Vol 86 (13) ◽

Author(s):

Huaduo Yan ◽

Xiuxiu Qin ◽

Li Wang ◽

Wenli Chen

Keyword(s):

Rna Helicase ◽

Rna Metabolism ◽

Rnase E ◽

Double Stranded Rna ◽

Content Type ◽

E Coli ◽

Dead Box ◽

Assembly Mechanism ◽

Rna Degradosome ◽

Pcc 7120

ABSTRACT At present, little is known about the RNA metabolism driven by the RNA degradosome in cyanobacteria. RNA helicase and enolase are the common components of the RNA degradosome in many bacteria. Here, we provide evidence that both enolase and the DEAD-box RNA helicase CrhB can interact with RNase E in Anabaena (Nostoc) sp. strain PCC 7120 (referred to here as PCC 7120). Furthermore, we found that the C-terminal domains of CrhB and AnaEno (enolase of PCC 7120) are required for the interaction, respectively. Moreover, their recognition motifs for AnaRne (RNase E of PCC 7120) turned out to be located in the N-terminal catalytic domain, which is obviously different from those identified previously in Proteobacteria. We also demonstrated in enzyme activity assays that CrhB can induce AnaRne to degrade double-stranded RNA with a 5′ tail. Furthermore, we investigated the localization of CrhB and AnaRne by green fluorescent protein (GFP) translation fusion in situ and found that they both localized in the center of the PCC 7120 cytoplasm. This localization pattern is also different from the membrane binding of RNase E and RhlB in Escherichia coli. Together with the previous identification of polynucleotide phosphorylase (PNPase) in PCC 7120, our results show that there is an RNA degradosome-like complex with a different assembly mechanism in cyanobacteria. IMPORTANCE In all domains of life, RNA turnover is important for gene regulation and quality control. The process of RNA metabolism is regulated by many RNA-processing enzymes and assistant proteins, where these proteins usually exist as complexes. However, there is little known about the RNA metabolism, as well as about the RNA degradation complex. In the present study, we described an RNA degradosome-like complex in cyanobacteria and revealed an assembly mechanism different from that of E. coli. Moreover, CrhB could help RNase E in Anabaena sp. strain PCC 7120 degrade double-stranded RNA with a 5′ tail. In addition, CrhB and AnaRne have similar cytoplasm localizations, in contrast to the membrane localization in E. coli.

Download Full-text

The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases

Biochemical Journal ◽

10.1042/bj3080839 ◽

1995 ◽

Vol 308 (3) ◽

pp. 839-846 ◽

Cited By ~ 15

Author(s):

J Sowden ◽

W Putt ◽

K Morrison ◽

R Beddington ◽

Y Edwards

Keyword(s):

Expression Profile ◽

Sequence Similarity ◽

Rna Helicase ◽

Chromosome 1 ◽

Rna Helicases ◽

High Sequence Similarity ◽

Dead Box ◽

Isolation And Characterization ◽

Helicase Gene ◽

The Dead

DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo.

Download Full-text

Genera specific distribution of DEAD-box RNA helicases in cyanobacteria

Microbial Genomics ◽

10.1099/mgen.0.000517 ◽

2021 ◽

Vol 7 (3) ◽

Cited By ~ 1

Author(s):

Denise S. Whitford ◽

Brendan T. Whitman ◽

George W. Owttrim

Keyword(s):

Phylogenetic Analysis ◽

Rna Helicase ◽

Evolutionary Divergence ◽

Sequence Motif ◽

Rna Helicases ◽

Homologous Proteins ◽

Content Type ◽

Conserved Sequence ◽

Link Type ◽

Dead Box

Although RNA helicases are essentially ubiquitous and perform roles in all stages of RNA metabolism, phylogenetic analysis of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase family in a single phylum has not been performed. Here, we performed a phylogenetic analysis on DEAD-box helicases from all currently available cyanobacterial genomes, comprising a total of 362 helicase protein sequences from 280 strains. DEAD-box helicases belonging to three distinct clades were observed. Two clades, the CsdA (cold shock DEAD-box A)-like and RhlE (RNA helicase E)-like helicases, cluster with the homologous proteins from Escherichia coli . The third clade, the CrhR (cyanobacterial RNA helicase Redox)-like helicases, is unique to cyanobacteria and characterized by a conserved sequence motif in the C-terminal extension. Restricted distribution is observed across cyanobacterial diversity with respect to both helicase type and strain. CrhR-like and CsdA-like helicases essentially never occur together, while RhlE always occurs with either a CrhR-like or CsdA-like helicase. CrhR-like and RhlE-like proteins occurred in filamentous cyanobacteria of the orders Nostocales , Oscillatoriales and Synechococcales . Similarly, CsdA- and RhlE-like proteins are restricted to unicellular cyanobacteria of the genera Cyanobium and Synechococcus . In addition, the unexpected occurrence of RhlE in two Synechococcus strains suggests recent acquisition and evolutionary divergence. This study, therefore, raises physiological and evolutionary questions as to why DEAD-box RNA helicases encoded in cyanobacterial lineages display restricted distributions, suggesting niches that require either CrhR or CsdA RNA helicase activity but not both. Extensive conservation of gene synteny surrounding the previously described rimO–crhR operon is also observed, indicating a role in the maintenance of photosynthesis. The analysis provides insights into the evolution, origin and dissemination of sequences within a single gene family to yield divergent functional roles.

Download Full-text

The DEAD box RNA helicase Ddx39a is essential for myocyte and lens development in zebrafish

10.1101/209650 ◽

2017 ◽

Author(s):

Linlin Zhang ◽

Beibei Li ◽

Yuxi Yang ◽

Ian C. Scott ◽

Xin Lou

Keyword(s):

Heart Development ◽

Rna Helicase ◽

Lens Fiber ◽

Rna Helicases ◽

Gene Encoding ◽

Animal Development ◽

Fiber Cells ◽

Dead Box ◽

The Dead ◽

Almost All

AbstractRNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease are largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a zebrafish mutant, which disrupts the gene encoding the RNA helicase DEAD-box 39a (ddx39a).Homozygous ddx39a mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Further investigation indicated that loss of ddx39a hindered mRNA splicing of members of the kmt2 gene family, leading to mis-regulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39a plays an essential role in establishment of proper epigenetic status during cell differentiation.

Download Full-text

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803

Journal of Bacteriology ◽

10.1128/jb.00267-16 ◽

2016 ◽

Vol 198 (15) ◽

pp. 2089-2099 ◽

Cited By ~ 7

Author(s):

Albert Remus R. Rosana ◽

Denise S. Whitford ◽

Richard P. Fahlman ◽

George W. Owttrim

Keyword(s):

Secondary Structure ◽

Thylakoid Membrane ◽

Rna Secondary Structure ◽

Mrna Translation ◽

Rna Helicase ◽

Rna Degradation ◽

Rna Helicases ◽

Content Type ◽

Rna Degradosome ◽

Pcc 6803

ABSTRACTThe cyanobacteriumSynechocystissp. strain PCC 6803 encodes a single DEAD box RNA helicase, CrhR, whose expression is tightly autoregulated in response to cold stress. Subcellular localization and proteomic analysis results indicate that CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with polysome and RNA degradosome components. Evidence is presented that either functional RNA helicase activity or a C-terminal localization signal was required for polysome but not thylakoid membrane localization. Polysome fractionation and runoff translation analysis results indicate that CrhR associates with actively translating polysomes. The data implicate a role for CrhR in translation or RNA degradation in the thylakoid region related to thylakoid biogenesis or stability, a role that is enhanced at low temperature. Furthermore, CrhR cosedimentation with polysome and RNA degradosome complexes links alteration of RNA secondary structure with a potential translation-RNA degradation complex inSynechocystis.IMPORTANCEThe interaction between mRNA translation and degradation is a major determinant controlling gene expression. Regulation of RNA function by alteration of secondary structure by RNA helicases performs crucial roles, not only in both of these processes but also in all aspects of RNA metabolism. Here, we provide evidence that the cyanobacterial RNA helicase CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with actively translating polysomes and RNA degradosome components. These findings link RNA helicase alteration of RNA secondary structure with translation and RNA degradation in prokaryotic systems and contribute to the data supporting the idea of the existence of a macromolecular machine catalyzing these reactions in prokaryotic systems, an association hitherto recognized only in archaea and eukarya.

Download Full-text

The Escherichia coli RNA degradosome: structure, function and relationship to other ribonucleolytic multienyzme complexes

Biochemical Society Transactions ◽

10.1042/bst0300150 ◽

2002 ◽

Vol 30 (2) ◽

pp. 150-155 ◽

Cited By ~ 117

Author(s):

A. J. Carpousis

Keyword(s):

Escherichia Coli ◽

Rna Binding ◽

Intrinsic Property ◽

Rna Helicase ◽

Rnase E ◽

E Coli ◽

Ribonucleolytic Activity ◽

Dead Box ◽

Rna Degradosome

mRNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript. The RNase E of Escherichia coli is a single-strand-specific endonuclease involved in the processing of rRNA and the degradation of mRNA. A nucleolytic multienzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E. Two other components are a 3′ exoribonuclease (polynucleotide phosphorylase, PNPase) and a DEAD-box RNA helicase (RNA helicase B, RhlB). RNase E is a large multidomain protein with N-terminal ribonucleolytic activity, an RNA-binding domain and a C-terminal ‘scaffold’ that binds PNPase, enolase and RhlB. RhlB by itself has little activity but is strongly stimulated by its interaction with RNase E. RhlB in vitro can facilitate the degradation of structured RNA by PNPase. Since the discovery of the RNA degradosome in E. coli, related complexes have been described in other organisms.

Download Full-text