The QKI-6 RNA Binding Protein Localizes with the MBP mRNAs in Stress Granules of Glial Cells

Background: The quaking viable (qk v) mouse has several developmental defects that result in rapid tremors in the hind limbs. The qkI gene expresses three major alternatively spliced mRNAs (5, 6 and 7 kb) that encode the QKI-5, QKI-6 and QKI-7 RNA binding proteins that differ in their C-terminal 30 amino acids. The QKI isoforms are known to regulate RNA metabolism within oligodendrocytes, however, little is known about their roles during cellular stress. Methodology/Principal Findings: In this study, we report an interaction between the QKI-6 isoform and a component of the RNA induced silencing complex (RISC), argonaute 2 (Ago2). We show in glial cells that QKI-6 co-localizes with Ago2 and the myelin basic protein mRNA in cytoplasmic stress granules. Conclusions: Our findings define the QKI isoforms as Ago2-interacting proteins. We also identify the QKI-6 isoform as a new component of stress granules in glial cells

Identification of Campylobacter jejuni genes contributing to acid adaptation by transcriptional profiling and genome-wide mutagenesis

In order to cause disease, the food- and waterborne pathogen Campylobacter jejuni must face the extreme acidity of the host stomach as well as cope with pH fluctuations in the intestine. In the present study, C. jejuni NCTC 11168 was grown under mildly acidic conditions mimicking those encountered in the intestine. The resulting transcriptional profiles revealed how this bacterium fine-tunes gene expression in response to acid stress. This adaptation involves the differential expression of respiratory pathways, the induction of genes for phosphate transport, and the repression of energy generation and intermediary metabolism genes. We also generated and screened a transposon-based mutant library to identify genes required for wild-type levels of growth under mildly acidic conditions. This screen highlighted the important role played by cell surface components (flagella, the outer membrane, capsular polysaccharides, and lipooligosaccharides) in the acid stress response of C. jejuni. Our data also revealed that a limited correlation exists between genes required for growth under acidic conditions and genes differentially expressed in response to acid. To gain a comprehensive picture of the acid stress response of C. jejuni, we merged transcriptional profiles obtained from acid-adapted cells and cells subjected to acid shock. Genes encoding the transcriptional regulator PerR and putative oxidoreductase subunits Cj0414 and Cj0415 were among the few up-regulated under both acid stress conditions. As a Cj0415 mutant was acid sensitive, it is likely that these genes are crucial to the acid stress response of C. jejuni and consequently are important for host colonization.Peer reviewedVeterinary Pathobiolog

The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation

We identify new mRNA targets for the QKI-6 RNA binding proteins using an unbiased approach. We show that AIP-1 mRNA is bound by QKI-6 within its 3′-UTR. This regulation is observed in oligodendrocytes and it is essential for oligodendrocyte process outgrowth

RNA interference is not affected by the absence of the QKI-6 isoform in U343 cells.

<p>(A) Control siRNA, siQKI-6 or siAgo2 were transfected in U343 cells and 24 hr later, the pMIR luciferase plasmid was co-transfected with the renilla luciferase vector and an siRNA that targets the pgl2 against firefly luciferase or a control siRNA. Thirty hours later, the cells were lysed and subjected to luciferase assays or immunoblotting analysis panel B. The firefly luciferase activity was normalized with renilla luciferase. (B) Cellular extracts from transfected U343 cells from panel (A) were immunoblotted with anti-Ago2 and anti-QKI antibodies. Immunoblotting against anti-Sam68 antibodies was used as a loading control. Absolute quantified levels of protein bands are presented underneath each blot and were performed using Scion-Image.</p

The endogenous QKI isoforms associate with Ago2.

<p>(A) U343 cell lysates were subjected to immunoprecipitations (IP) with control immunoglobulin G (IgG), anti-QKI-5, -QKI-6 and -QKI-7 antibodies. The bound proteins were separated by SDS-PAGE and immunoblotted (IB) with anti-Ago2 antibodies. (B) U343 cell lysates were treated with 1 mg/ml RNase A, 2 U/100 µl RNase V1 or RNase inhibitor as indicated at 37°C for 1 hr and subjected to immunoprecipitation with the anti-QKI-6 antibody. The proteins were separated by SDS-PAGE and immunoblotted with anti-Ago-2 antibodies as indicated (upper panel). The activity of the RNases and the RNase inhibitor was verified by agarose gel electrophoresis with 10 µg of total RNA (lower panel).</p

DCP-1 P bodies are devoid of QKI-7.

<p>Myc-QKI-7 and Flag-DCP1 were co-transfected in HEK293 cells. Thirty hours later, the cells were left untreated or treated with 0.5 mM As₂O₃ for 30 min. The cells were fixed, permeabilized and immunostained with rabbit anti-Myc antibody and mouse anti-Flag antibody followed by goat anti-rabbit Alexa523 (red) and goat anti-mouse Alexa 488 (green). The nuclei were counter-stained with DAPI. The scale bar represents 10 µm.</p

QKI-6 and QKI-7 isoforms co-localize with Ago2 in cytoplasmic granules in glial cells.

<p>(A) U343 cells were untreated or treated with 0.5 mM arsenic oxide (As₂O₃) for 45 min. The cells were fixed, permeabilized and immunostained with rabbit anti-QKI-5, -6 and -7 antibodies and mouse anti-Ago2 antibodies followed by secondary goat anti-rabbit Alexa 488 (green) and goat anti-mouse Alexa 523 (red) antibodies. The nuclei were counter-stained with DAPI. The scale bar represents 5 µm. (B) The quantification of the co-localization between the QKI isoforms and Ago2 expressed as a percentage is shown. (C) Primary rat oligodendrocytes were untreated or treated with 0.5 mM As₂O₃ for 45 min and analyzed as in panel (A). (D) The quantification was performed as in panel (B).</p

Mapping the domains required for the QKI-6/Ago2 interaction.

<p>(A) A schematic illustration of the QKI-6 protein showing its regions and its amino acid numbering. Expression vectors encoding GFP-QKI-6 and truncation mutants thereof were transfected in HEK293 cells. The transfected cells were lysed and the cell lysates were subjected to immunoprecipitation with the anti-GFP antibody and the bound proteins separated by SDS-PAGE. The presence of Ago2 was monitored by using anti-Ago2 antibodies as indicated (left panel). Extracts before immunoprecipitation were separated by SDS-PAGE and immunoblotted with anti-GFP antibodies to confirm equivalent expression. The molecular mass markers are shown on the left in kDa. (B) A schematic illustration of Ago2 is shown with its conserved domains and the numbering of its residues. The GFP-QKI-6 expression plasmid was cotransfected with myc-tagged full-length Ago2 or truncation mutants in HEK293 cells, as indicated. Twenty four hours after transfection, the cells were lysed and cell lysates were subjected to immunoprecipitation with anti-Myc antibodies followed by immunoblotting with anti-GFP antibodies. The migration of GFP-QKI-6 is shown (left panel), while the expression of the myc-Ago2 proteins is shown in the right panel. The asterisks denote non-specific proteins recognized by the anti-myc antibodies. The molecular mass markers are shown on the left in kDa.</p