Vanessa Khemici

The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers

In Escherichia coli, REP‐stabilizers are structural elements in polycistronic messages that protect 5′‐proximal cistrons from 3′→5′ exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP‐stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP‐stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD‐box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP‐stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP‐stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.

The RNase E of Escherichia coli is a membrane-binding protein.

RNase E is an essential endoribonuclease involved in RNA processing and mRNA degradation. The N‐terminal half of the protein encompasses the catalytic domain; the C‐terminal half is the scaffold for the assembly of the multienzyme RNA degradosome. Here we identify and characterize ‘segment‐A’, an element in the beginning of the non‐catalytic region of RNase E that is required for membrane binding. We demonstrate in vitro that an oligopeptide corresponding to segment‐A has the propensity to form an amphipathic α‐helix and that it avidly binds to protein‐free phospholipid vesicles. We demonstrate in vitro and in vivo that disruption of segment‐A in full‐length RNase E abolishes membrane binding. Taken together, our results show that segment‐A is necessary and sufficient for RNase E binding to membranes. Strains in which segment‐A has been disrupted grow slowly. Since in vitro experiments show that phospholipid binding does not affect the ribonuclease activity of RNase E, the slow‐growth phenotype might arise from a defect involving processes such as accessibility to substrates or interactions with other membrane‐bound machinery. This is the first report demonstrating that RNase E is a membrane‐binding protein and that its localization to the inner cytoplasmic membrane is important for normal cell growth.

The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE.

The non‐catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD‐box RNA helicase, RhlB. We show that three other DEAD‐box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791–843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP‐dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.

Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, pIRL, in the left end (IRL). Circle junction formation creates a strong promoter, pjunc, with a −35 sequence located in the right end and the −10 sequence in the left. pjunc assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in pjunc disassembly and a return to the low pIRL‐ driven transposase levels. We demonstrate that pjunc plays an important role in regulating IS911 transposition. Inactivation of pjunc strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess pjunc equivalents, underlining that the regulatory mechanisms adopted to fine‐tune transposition may be different.

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling.

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.

Co-immunopurification of multiprotein complexes containing RNA-degrading enzymes.

Co-immunopurification is a classical technique in which antiserum raised against a specific protein is used to purify a multiprotein complex. We describe work from our laboratory in which co-immunopurification was used to characterize the RNA degradosome of Escherichia coli, a multiprotein complex involved in RNA processing and mRNA degradation. Polyclonal rabbit antibodies raised against either RNase E or PNPase, two RNA degrading enzymes in the RNA degradosome, were used in co-immunopurification experiments aimed at studying the assembly of the RNA degradosome and mapping protein-protein interactions within the complex. In E. coli, this method has been largely supplanted by approaches in which proteins are engineered to contain tags that interact with commercially available antibodies. Nevertheless, we believe that the method described here is valid for the study of bacteria in which the genetic engineering needed to introduce tagged proteins is difficult or nonexistent. As an example, we briefly discuss ongoing work in our laboratory on the characterization of RNase E in the psychrotolerant bacterium Pseudoalteromonas haloplanktis.

Chapter 10 Assaying DEAD‐box RNA Helicases and Their Role in mRNA Degradation in Escherichia coli

The DEAD-box RNA helicases are a ubiquitous family of enzymes involved in processes that include RNA splicing, ribosome biogenesis, and mRNA degradation. In general, these enzymes help to unwind short stretches of double-stranded RNA in processes that involve the remodeling of RNA structure or of ribonucleoprotein complexes. Here we describe work from our laboratory on the characterization of the RhlB of Escherichia coli, a DEAD-box RNA helicase that is part of a multienzyme complex known as the RNA degradosome. RhlB interacts physically and functionally with RNase E and polynucleotide phosphorylase (PNPase), two other components of the RNA degradosome. We describe enzyme assays that demonstrated that the interaction between RhlB and RNase E is necessary for the ATPase and RNA unwinding activities of RhlB. We also describe an mRNA degradation assay that showed that RhlB facilitates the degradation of structured mRNA by PNPase. These assays are discussed in the context of how they have contributed to our understanding of the function of RhlB in mRNA degradation.