Sequence similarity between stereocilin and otoancorin points to a unified mechanism for mechanotransduction in the mammalian inner ear

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Interaction between hair cells and acellular gels of the mammalian inner ear, the tectorial and otoconial membranes, is crucial for mechanoreception. Recently, otoancorin was suggested to be a mediator of gel attachment to nonsensory cells, but the molecular components of the interface between gels and sensory cells remain to be identified. Hypothesis We report that the inner ear protein stereocilin is related in sequence to otoancorin and, based on its localisation and predicted GPI-anchoring, may mediate attachment of the tectorial and otoconial membranes to sensory hair bundles. Testing It is expected that antibodies directed against stereocilin would specifically label sites of contact between sensory hair cells and tectorial/otoconial membranes of the inner ear. Implications Our findings support a unified molecular mechanism for mechanotransduction, with stereocilin and otoancorin defining a new protein family responsible for the attachment of acellular gels to both sensory and nonsensory cells of the inner ear.Published versio

The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation

BACKGROUND: Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown. RESULTS: By exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments. CONCLUSION: These findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes

6S RNA regulation of relA alters ppGpp levels in early stationary phase

6S RNA is a small, non-coding RNA that interacts directly with σ70-RNA polymerase and regulates transcription at many σ70-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ70-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σS activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression

An Integrated Approach for Finding Overlooked Genes in Shigella

Background: The completion of numerous genome sequences introduced an era of whole-genome study. However, many genes are missed during genome annotation, including small RNAs (sRNAs) and small open reading frames (sORFs). In order to improve genome annotation, we aimed to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery. Methodology/Principal Findings: We identified 64 sRNAs in Shigella, which were experimentally validated in other bacteria based on sequence conservation. We employed computer-based and tiling array-based methods to search for sRNAs, followed by RT-PCR and northern blots, to identify nine sRNAs in Shigella flexneri strain 301 (Sf301) and 256 regions containing possible sRNA genes. We found 29 candidate sORFs using bioinformatic prediction, array hybridization and RT-PCR verification. We experimentally validated 557 (57.9%) DOOR operon predictions in the chromosomes of Sf301 and 46 (76.7%) in virulence plasmid.We found 40 additional co-expressed gene pairs that were not predicted by DOOR. Conclusions/Significance: We provide an updated and comprehensive annotation of the Shigella genome. Our study increased the expected numbers of sORFs and sRNAs, which will impact on future functional genomics and proteomics studies. Our method can be used for large scale reannotation of sRNAs and sORFs in any microbe with a known genom

Egg's ZP3 Structure Speaks Volumes

Binding of mammalian sperm to eggs depends in part on ZP3, a glycoprotein in the egg's extracellular coat, the zona pellucida. In this issue, Han et al. (2010) describe the structure of an avian ZP3 homolog, providing insights into ZP3 processing and polymerization and the roles of the ZP3 polypeptide and its carbohydrate in sperm binding