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Structural Characterization of nc886, a Cellular Non‐coding RNA Regulator of the Antiviral Kinase PKR
Author(s) -
Calderon Brenda,
Conn Graeme
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.711.7
Subject(s) - protein kinase r , rna , biology , eif 2 kinase , antiviral protein , microbiology and biotechnology , kinase , viral replication , regulator , protein kinase a , virology , biochemistry , virus , gene , mitogen activated protein kinase kinase , cyclin dependent kinase 2
The double‐stranded (ds)RNA‐activated protein kinase (PKR) senses dsRNA produced during viral replication and acts as the first line of defense in the innate immune response to viral infection. Activated PKR phosphorylates the eukaryotic initiation factor 2 (eIF2), halting protein synthesis and viral replication. How PKR basal activity is regulated in the absence of viral infection is unknown. Recently, the cellular non‐coding RNA 886 (nc886) was discovered and proposed to act as a novel, endogenous regulator of PKR. We hypothesize that nc886 regulates PKR by binding to the monomeric form and preventing the dimerization step of activation. This maintains PKR in an inactive state that is poised for a rapid response to viral infection. Our goal is to define the molecular mechanism of nc886‐mediated PKR inhibition using RNA structure probing and mutational analyses combined with binding and activity assays. We show that nc886 RNA exists as two stable, structurally and functionally distinct conformations. Ribonuclease digestion reveals that the two conformers differ in their apical region, while the terminal stem portion of the RNA structure is conserved. The two conformers have strikingly different binding affinities for PKR, but both are functional inhibitors of PKR in in vitro kinase assays. By delineating the structural basis for PKR interaction with the different nc886 conformers, we will uncover the structural motifs that control PKR inhibition and determine how PKR is poised to act in the antiviral response. Source of Research Support: NIH R21‐AI097803 and 5T32GM008367