Escherichia coli RNase E is an essential enzyme that forms multicomponent ribonucleolytic complexes known as "RNA degradosomes." These complexes consist of four major components: RNase E, PNPase, RhlB RNA helicase, and enolase. However, the role of enolase in the RNase E/degradosome is not understood. Here, we report that presence of enolase in the RNase E/degradosome under anaerobic conditions regulates cell morphology, resulting in E. coli MG1655 cell filamentation. Under anaerobic conditions, enolase bound to the RNase E/degradosome stabilizes the small RNA (sRNA) DicF, i.e., the inhibitor of the cell division gene ftsZ, through chaperon protein Hfq-dependent regulation. RNase E/enolase distribution changes from membraneassociated patterns under aerobic to diffuse patterns under anaerobic conditions. When the enolase-RNase E/degradosome interaction is disrupted, the anaerobically induced characteristics disappear. We provide a mechanism by which E. coli uses enolase-bound degradosomes to switch from rod-shaped to filamentous form in response to anaerobiosis by regulating RNase E subcellular distribution, RNase E enzymatic activity, and the stability of the sRNA DicF required for the filamentous transition. In contrast to E. coli nonpathogenic strains, pathogenic E. coli strains predominantly have multiple copies of sRNA DicF in their genomes, with cell filamentation previously being linked to bacterial pathogenesis. Our data suggest a mechanism for bacterial cell filamentation during infection under anaerobic conditions. RNase E | RNA decay | protein subcellular distribution | anaerobic conditions | cell shape P osttranscriptional regulation of RNAs is an important molecular mechanism for controlling gene expression, requiring various ribonucleases (RNases), including RNase E, which is an essential single-stranded endo-RNase involved in RNA processing and decay (1). RNase E has N-terminal catalytic and C-terminal scaffolding domains (2), with the latter responsible for assembling multicomponent ribonucleolytic complexes termed "RNA degradosomes." Degradosomes consist of RNase E, PNPase 3′→5′ exoribonuclease, RhlB RNA helicase, and the glycolytic enzyme enolase (3, 4). Therefore, they can act on RNA internally (by RNase E) and/or externally (by PNPase) to catalyze the degradation of RNA into short fragments. Immunogold electron microscopy has shown that degradosomes exist in vivo and are tethered to the cytoplasmic membrane through the N-terminal region of RNase E (5). Binding of the N-terminal catalytic domain (amino acids 1-499) to the membrane stabilizes protein structure and increases both RNA cleavage activity and substrate affinity (6). Global analyses of aerobic Escherichia coli RNA degradosome functioning using DNA microarrays showed that decay of some mRNAs in vivo depends on the action of assembled degradosomes, whereas other mRNAs are impacted by degradosome proteins functioning independently of the complex (7-9). Some minor components of the degradosome, such as the inhibitors of RNase E, RraA and ...

RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K d ) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60°C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay.R Nase E has an essential role in RNA processing and decay in Escherichia coli (1), and it has been postulated that its homologs in other bacteria may have similar functions (2-5). E. coli RNase E, encoded by the rne gene, is a large, multidomain protein of 1,061 amino acids with several functionally distinct regions (1, 6, 7). The first 395 amino acids of E. coli RNase E confer a single-strand-specific endonuclease activity and are highly evolutionarily conserved (8-10). The crystal structure of the truncated RNase E polypeptide [Protein Data Bank (PDB) ID code: 2C4R], determined at 2.9 Å, revealed a structurally defined catalytic domain (designated the "large" domain, amino acids 1-400), followed by a Zn-link spacer (amino acids 400-415) and a "small" domain (amino acids 415-529) that serves as a dimerization interface (11). The large domain consists of several subdomains including the 5′-sensor as well as subdomains structurally similar to protein folds found in S1, DNase I, and RNase H. The C-terminal noncatalytic domain of E. coli RNase E also contains functionally important regions including an arginine-rich region (amino acids 597-684) involved in RNA-binding (12) and a "scaffold" region (amino acids 650-1061) for RNA degradosome a...

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporal-spatial manner. Zinc finger-containing transcription factors have been implicated as critical regulators of multiple cardiac-expressed genes, and are thought to be important for human heart development and diseases. Here, we have identified and characterized a novel zinc finger gene named ZNF418 from a human embryo heart cDNA library. The gene spans 13.5 kb on chromosome 19q13.43 encompassing six exons, and transcribes a 3.7-kb mRNA that encodes a protein with 676 amino acid residues. The predicted protein contains a KRAB-A box and 17 tandem C2H2 type zinc finger motifs. Northern blot analysis indicates that ZNF418 is expressed in multiple fetal and adult tissues, but is expressed at higher levels in the heart. Reporter gene assays show that ZNF418 is a transcriptional repressor, and the KRAB motif of ZNF418 represents the basal repressive domain. Overexpression of ZNF418 in COS-7 cells inhibits the transcriptional activity of SRE and AP-1 which may be silenced by siRNA. These results suggest that ZNF418 is a member of the zincfinger transcription factor family and may act as a negative regulator in MAPK signaling pathway.

Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5′ or 3′ half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon. IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

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