The objective of this application is to uncover roles for RNA structure in regulating biological activity of the RNA-activated protein kinase, PKR. Viral and cellular RNAs fold into diverse secondary and tertiary structures and interact with proteins to alter the innate immune response. A key player in innate immunity is the interferon-induced RNA-activated protein kinase, PKR. The major activator of PKR in vivo has been proposed to be long dsRNA (>33 bp), which can bridge two PKR monomers and increase their effective concentration. Interaction with dsRNA also relieves an inhibitory interaction in the kinase. Subsequently, PKR carries out trans-autophosphorylation that activates it to phosphorylate eIF21, which inhibits the initiation of translation. Despite the obvious ability of dsRNA to activate PKR in vitro, it remains unclear whether such RNAs are major activators of PKR in vivo. The central hypothesis of this proposal is that regulation of PKR in vivo is mediated by novel RNA motifs with unconventional structures. In particular, evidence is presented that ssRNA activates PKR in a 5'-triphosphate-dependent fashion, which is blocked by cellular 5'-end signatures of 7mG and monophosphate. Because a 5'-triphosphate occurs on many pathogenic RNAs, this suggests a novel pathogen-associated molecular pattern (PAMP) that is recognized by PKR. Moreover, additional evidence suggests that self RNA is also distinguished by internal nucleoside modifications, which are shown to abrogate PKR activation. The central aims of the proposal are as follows: 1.) Determine the mechanism by which ssRNA activates PKR in a 5'-triphosphate-specific fashion, often with the assistance of short stem-loops. Develop a molecular model for interaction of the 5'-triphosphate and short stem-loop with PKR, as well as a mechanistic framework for activation. 2.) Establish roles for posttranscriptional RNA modifications and non-Watson-Crick motifs in modulating PKR activation. Identify patterns of modifications and non-Watson-Crick motifs that allow cellular RNAs to evade PKR activation. 3.) Determine roles for short viral RNA secondary structures and globular tertiary motifs in modulating PKR activation. Test if certain viral secondary structure RNAs dimerize to form PKR-activating motifs, while globular RNA tertiary structures fold to mask PKR-activating RNA secondary structures. 4.) Identify viral and endogenous RNAs that regulate PKR in vivo. Use cross-linking and immunoprecipitation (CLIP) technologies with (and without) vesicular stomatitis virus transfections and infections.
These Specific Aims will be accomplished by a variety of biochemical and molecular biology techniques including RNA and protein mutagenesis, in vitro and in vivo PKR and eIF21 activation assays, kinetics and thermodynamic measurements, and CLIP experiments. Binding assays will be conducted by fluorescence polarization and ITC;protein dimerization will be monitored by pull-down assays, crosslinking, and analytical ultracentrifugation;RNA-protein interactions will be assayed by chemical crosslinking and mutagenesis;and RNA tertiary structure will be assessed by native gel electrophoresis and structure mapping.

Public Health Relevance

Innate immunity offers a host early protection from foreign organisms and viruses, and the protein PKR is an important part of this response in humans. This proposal aims to understand how novel molecular patterns in pathogenic RNA are recognized by PKR as different from self RNA, which cause the innate immune response to be initiated. Viral RNAs with potentially pathogenic patterns from human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) will be studied.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058709-13
Application #
8231406
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Preusch, Peter C
Project Start
1999-01-01
Project End
2013-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
13
Fiscal Year
2012
Total Cost
$277,858
Indirect Cost
$85,252
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Nallagatla, Subba Rao; Jones, Christie N; Ghosh, Saikat Kumar B et al. (2013) Native tertiary structure and nucleoside modifications suppress tRNA's intrinsic ability to activate the innate immune sensor PKR. PLoS One 8:e57905
Nallagatla, Subba Rao; Toroney, Rebecca; Bevilacqua, Philip C (2011) Regulation of innate immunity through RNA structure and the protein kinase PKR. Curr Opin Struct Biol 21:119-27
Heinicke, Laurie A; Nallagatla, Subba Rao; Hull, Chelsea M et al. (2011) RNA helical imperfections regulate activation of the protein kinase PKR: effects of bulge position, size, and geometry. RNA 17:957-66
Chadalavada, Durga M; Gratton, Elizabeth A; Bevilacqua, Philip C (2010) The human HDV-like CPEB3 ribozyme is intrinsically fast-reacting. Biochemistry 49:5321-30
Toroney, Rebecca; Nallagatla, Subba Rao; Boyer, Joshua A et al. (2010) Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A. J Mol Biol 400:393-412
Veeraraghavan, Narayanan; Bevilacqua, Philip C; Hammes-Schiffer, Sharon (2010) Long-distance communication in the HDV ribozyme: insights from molecular dynamics and experiments. J Mol Biol 402:278-91
Anderson, Bart R; Muramatsu, Hiromi; Nallagatla, Subba R et al. (2010) Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 38:5884-92
McGraw, Adam P; Mokdad, Ali; Major, Francois et al. (2009) Molecular basis of TRAP-5'SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism. RNA 15:55-66
Heinicke, Laurie A; Wong, C Jason; Lary, Jeffrey et al. (2009) RNA dimerization promotes PKR dimerization and activation. J Mol Biol 390:319-38
Nallagatla, Subba Rao; Toroney, Rebecca; Bevilacqua, Philip C (2008) A brilliant disguise for self RNA: 5'-end and internal modifications of primary transcripts suppress elements of innate immunity. RNA Biol 5:140-4

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