The E. coli RhlE RNA helicase regulates the function of related RNA helicases during ribosome assembly

Abstract

Escherichia coli contains five members of the DEAD-box RNA helicase family, a ubiquitous class of proteins characterized by their ability to unwind RNA duplexes. Although four of these proteins have been implicated in RNA turnover or ribosome biogenesis, no cellular function for the RhlE DEAD-box protein has been described as yet. During an analysis of the cold-sensitive growth defect of a strain lacking the DeaD/CsdA RNA helicase, rhlE plasmids were identified from a chromosomal library as multicopy suppressors of the growth defect. Remarkably, when tested for allele specificity, RhlE overproduction was found to exacerbate the cold-sensitive growth defect of a strain that lacks the SrmB RNA helicase. Moreover, the absence of RhlE exacerbated or alleviated the cold-sensitive defect of deaD or srmB strains, respectively. Primer extension and ribosome analysis indicated that RhlE regulates the accumulation of immature ribosomal RNA or ribosome precursors when deaD or srmB strains are grown at low temperatures. By using an epitope-tagged version of RhlE, the majority of RhlE in cell extracts was found to cosediment with ribosome-containing fractions. Since both DeaD and SrmB have been recently shown to function in ribosome assembly, these findings suggests that rhlE genetically interacts with srmB and deaD to modulate their function during ribosome maturation. On the basis of the available evidence, I propose that RhlE is a novel ribosome assembly factor, which plays a role in the interconversion of ribosomal RNA-folding intermediates that are further processed by DeaD or SrmB during ribosome maturation.

FIGURE 1.

Effect of RhlE overexpression on the cold-sensitive growth of ΔdeaD and ΔsrmB strains. (A) Suppression of the ΔdeaD cold-sensitive growth defect by RhlE overexpression. A control plasmid (pACYC184) or a RhlE plasmid (pCJ1078) were transformed into MG1655ΔdeaD. The transformed strains were streaked on a LB-agar-chloramphenicol plate, followed by incubation at 20°C. (B) MG1655 or the indicated derivative strains were transformed with pACYC184 or pCJ1078 and grown in LB supplemented with chloramphenicol at 16°C or 20°C. The strain doubling time was derived from measurements of cell culture density versus time for three to four independent cultures. The mean cell doubling time and standard deviation are indicated. (n.d.) Not determined. (C) RhlE overexpression exacerbates the ΔsrmB cold-sensitive growth defect. MG1655ΔsrmB was transformed with either pACYC184 or pCJ1078, streaked on a LB-agar-chloramphenicol plate, and grown at 16°C.

FIGURE 2.

Growth of wild-type and helicase-deficient strains at low temperatures. (A) Strain MG1655 (wild type) and isogenic derivatives containing ΔrhlE, ΔdeaD, or ΔdeaDΔrhlE mutations were streaked on LB-agar plates and incubated at 20°C. (B) MG1655 and isogenic derivatives containing ΔrhlE, ΔsrmB, or ΔsrmBΔrhlE deletions were streaked on LB-agar plates and incubated at 16°C.

FIGURE 3.

23S rRNA processing defects in helicase-deficient strains. RNA was extracted from the indicated strains, annealed with a labeled oligonucleotide probe (5′-CCTTCATCGCCTCTGACTGCC-3′) that hybridizes to 23S rRNA, reverse transcribed, and fractionated on a denaturing gel. The reverse transcription products that correspond to the mature 5′ end (M), and to a precursor containing seven additional nucleotides (P), are indicated. An intermediate product corresponding to the +3 product can also be visualized. The assignment of the precursor and mature products are based on additional experiments (data not shown). The ratios of the +7 precursor to mature RNA were quantified by phosphorimaging. (A) RNA was analyzed from isogenic MG1655 derivatives grown at 16°C: lane 1, wild type; lane 2, ΔrhlE; lane 3, ΔsrmB; lane 4, ΔsrmBΔrhlE. (B) RNA was analyzed from MG1655 derivatives grown at 22°C: lane 1, wild type; lane 2, ΔrhlE; lane 3, ΔdeaD; lane 4, ΔdeaDΔrhlE.

FIGURE 4.

Ribosome profiles of mutant strains and RNA analysis. Cell lysates were fractionated by ultracentrifugation on a 14%–32% sucrose density gradient and ribosomal profiles were generated. (A) Ribosomal profiles derived from MG1655ΔdeaD (left) or MG1655ΔdeaDΔrhlE (right) grown at 22°C. Sedimentation is from right to left. The positions of the 70S ribosome, the 50S and 30S subunits, and a 40S precursor that accumulates in these strains are shown. The designation of these peaks is based on parallel ultracentrifugation studies on a wild-type strain in which 30S, 50S, and 70S peaks were well separated (data not shown). The numbers below the sedimentation values correspond to 12 fractions, starting at the 70S ribosomal peak and ending at the 30S subunit peak, which were collected for RNA analysis (see below). (B) RNA was extracted from the fractions underlined in A and analyzed by primer extension using a 23S rRNA probe (Materials and Methods). The migration of the mature (M) and two prominent precursors (+3) and (+7) are indicated. The abundance of the mature and precursor products were quantified by phosphorimaging, and the percentage of RNA that contains a mature end is denoted. (C) Ribosomal profiles derived from MG1655ΔsrmB (left) and MG1655ΔsrmBΔrhlE strains (right) grown at 16°C and analyzed as described in A.

FIGURE 5.

Association of RhlE with ribosomes. A Flag-tagged RhlE plasmid construct, pCJ1086, was transformed into MG1655ΔrhlE (A) or MG1655ΔrhlEΔdeaD (B). Transformed cells were grown in LB-ampicillin at 20°C, induced with arabinose, and harvested by centrifugation. To better discriminate between 70S ribosomes and polysomes, an extended sucrose concentration range (14%–40%) was used, as compared with 14%–32% used in the previous set of experiments, and 18 2-mL fractions were collected. (Top) The absorbance of each fraction at 260 nM was measured. The positions of the 70S ribosomes and the ribosomal subunits are indicated. (Bottom) Equal volumes of the fractions were used for Western blot analysis to detect Flag-tagged RhlE. The migration of Flag-RhlE is indicated by an arrow, and the positions of molecular weight markers are indicated.

FIGURE 6.

Proposed function of RhlE in ribosome assembly. RhlE is proposed to play a role in the interconversion of two sets of 23S rRNA conformational forms within pre-50S ribosomes that arise during the ribosome maturation process. Conformational form (A) denotes substrates that require DeaD for further processing, whereas form (B) denotes substrates that require SrmB. The absence of RhlE shifts the population toward form (A), increasing the dependence for DeaD, and alleviating the requirement for SrmB. The overproduction of RhlE has the opposite effect.