| Various cellular responses act as defense mechanisms against external stimuli, such as viral infections, heat shock, redox stress, endoplasmic reticulum(ER) stress, and UV irradiation. Cellular responses include innate immunity, the formation of stress granules(SGs), and autophagy.Innate immunity, one of the best-characterized responses, employs innate sensors to recognize invaders and activate downstream signaling pathways. Typically, viral infections are sensed by membrane-bound Toll like receptors(TLRs) or cytoplasmic RIG-I-like receptors(RLRs), including retinoic acid-inducible gene-I(RIG-I) and melanoma differentiation-associated gene 5(MDA5).The formation of stress granules(SGs) is another important cellular response to stress. In latent cells, cytoplasmic m RNAs are controlled via the regulation of m RNA translation, stability, and subcellular location. When translation initiation is impaired under stress, the stalled m RNPs accumulate at cytoplasmic foci(i.e., SGs).Autophagy is an evolutionarily conserved process which is involved in maintaining cytoplasmic homeostasis by degrading damaged organelles or misfolded proteins. The autophagic cascade is initiated by the engulfment of cytoplasmic cargoes by an autophagosome, which then fuses with a late endosome to form the autolysosome, exposing the inner compartment to lysosomal hydrolases for degradation.We had identified that RNA helicase A(RHA) could interact with PKR, an initiation factor of SGs, and demonstrated that the innate immune adaptor IFN-? promoter stimulator(IPS-1) was involved in the formation of ds RNA-induced SGs, indicating a connection between various stress responses. In this study, we further explored whether there was a relation among the three stress responses using another type of stress, i.e., carbonyl cyanide m-chlorophenylhydrazone(CCCP), a mitochondrial protonophore, which could induced formation of SG and autophagy. Additionally, we further explored the interaction of RHA and PKR and their influence on the formation of SG.Part?: Melanoma differentiation-associated gene 5 is involved in the induction of stress granules and autophagy by protonophore CCCPObjective: To explore the role of different innate immune proteins in the formation of SGs and autophagy induced by CCCPMethods: 1. The innate immune molecules PKR, IPS-1, RIG-I, and MDA5 were silenced with small interfering RNA(si PKR, si IPS-1, si RIG-I, si MDA5) in A549 and He La cells respectively, and the efficacy of si RNAs was validated by Western blotting(WB). 2. Cells were treated with CCCP to trigger the formation of SG. Immunofluorescence staining(IF) was used to detect SG formation using TIA-1, G3BP1 and TIAR. 2. A549 and He La cells were transfected with or without si MDA5, and then treated with CCCP. Activation of e IF2? was determined by Western blotting. 3. He La and A549 cells were mock-stressed or stressed by CCCP, poly(I:C), hippuristanol, or heat-shock. IF was used to detect SG formation stained by TIA-1 and G3BP1 and activation of MDA5. 4. MDA5-sufficient and MDA5-deficient cells were treated with CCCP, rapamycin, and starvation. Western blotting and IF were used to detect the transformation of LC3-I to LC3-II. Rapamycin and starvation treated cells were examined as control. 5. A549 and He La cells were silenced with si RNA against G3BP1, and were treatedwith CCCP. Western blotting and IF were used to detect the transformation of LC3-I to LC3-II.Results: 1. WB and IF results showed that the si RNAs(si PKR, si IPS-1, si RIG-I, and si MDA5) could significantly reduce the level of targeted proteins. The percentage of SG-positive cells in si MDA5-transfected A549 cells was markedly lower than that observed in si NC-transfected cells, while the percentages of SG-positive cells with depleted RIG-I, PKR, or IPS-1 were comparable to those of control cells. 2. The phosphorylation levels of e IF2? in all CCCP-stimulated cells were significantly higher than those of mock-treated cells. And the phosphorylation levels of e IF2? were comparable for MDA5 knockdown and control cells 3. TIA-1 and G3BP1 proteins were translocated to cytoplasmic SG foci under all stress treatments, while MDA5 was not recruited to these SGs. 4. The ratios of LC3-II to actin were significantly higher in cells treated with CCCP, rapamycin, or starvation than in mock-treated cells. MDA5 knockdown led to a significant decrease in LC3-II in CCCP-treated cells, but had no or minimal effect on the levels of LC3-II in cells treated with rapamycin or starvation. 5. The ratio of LC3-II to actin in si G3BP1-transfected cells was significantly lower than that in si NC-transfected cellsConclusion: 1. MDA5 played an important role in SG formation induced by CCCP. 2. MDA5 did not have a direct effect on e IF2? phosphorylation and functioned downstream of e IF2? phosphorylation. 3. MDA5 was not a component of SGs induced by CCCP. 4. MDA5 was involved in the autophagy process triggered by CCCP. 5. Autophagy triggered by CCCP might rely on SG formation. In summary, our work uncovered a novel role of the innate molecule MDA5 in SG formation and autophagy induced by CCCP, providing further evidence for the correlation between different stress responses.Part II: Role of RNA helicase A in ds RNA-induced cytoplasmic granulesObjective: To further explore the domains that mediate RHA and PKR interaction and the relation among RHA, PKR and SG using immunofluorescence assay.Methods: 1. Cells were mock or stimulated with poly(I:C). RHA and PKR co-localization was determined by IF using antibodies against RHA and PKR. 2. RHAN, RHAM and RHAC c DNA were cloned into p EGFP-C1 respectively to obtain GFP-tagged truncated RHA segment. m Cherry fragment was amplified by PCR, digested with indicated enzymes, and then inserted into p SG5, p SG5-PKRN or p SG5-PKRC. Cells, transiently transfected with these plasmids, were treated with mock or poly(I:C) to identify which segment of RHA or PKR was essential for cytoplasmic granules formation. p EGFP-C1 and p SG5-m Cherry were examined as control. 3. Cells were transfected with GFP-RHAN and m Cherry-PKRN vectors, and then mock or stimulated with poly(I:C). Their co-localization was determined by IF. 4. Cells were mock or stimulated with poly(I:C). RHA/TIA-1, RHA/G3BP1 and PKR/TIA-1, PKR/G3BP1 co-localization were determined by IF using antibodies against RHA, PKR, TIA-1, or G3BP1. 5. PKR and RHA were silenced with small interfering RNA(si PKR and si RHA) in cells respectively, and then treated with poly(I:C). IF was used to detect their expression using RHA, PKR, TIA-1, and G3BP1 antibodies, and the efficacy of si RNAs was validated by WB.Results: 1. RHA protein was transported into cytoplasmic and colocalized with PKR granules under poly(I:C) treatment. 2. The N terminus of RHA and PKR were essential for its assembly in to cytoplasmic granules under poly(I:C) treatment. 3. The N terminus of RHA and PKR showed a good co-localization under poly(I:C) treatment. 4. TIA-1 granules were overlapped with both RHA and PKR granules, but the G3BP1-RHA or G3BP1-PKR showed an absence of well correlation. 5. In PKR knockdown cells, the percentages of cells containing PKR, RHA, TIA-1 or G3BP1 granules were dramatically reduced; RHA depletion only significantly downregulated the percentages of cells containing RHA or TIA-1.Conclusion: 1. RHA could colocalize with PKR in poly(I:C) treated cells. 2. The N terminus of PKR or RHA were sufficient for their recruitment into cytoplasmic granules and co-localization. 3. RHA, PKR and TIA-1 were colocalized in ds RNA-induced granules, but the granules might not be conventional stress granules. 4. RHA played a different role from PKR in the formation of ds RNA-induced granules.In summary, our work further demonstrated the interaction between PKR and RHA, and uncovered a new role of RHA in cell physiology. |
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