RNA helical imperfections regulate activation of the protein kinase PKR: Effects of bulge position, size, and geometry

Heinicke, Laurie A.; Nallagatla, Subba Rao; Hull, Chelsea M.; Bevilacqua, Philip C.

doi:10.1261/rna.2636911

Cited by 18 publications

(20 citation statements)

References 38 publications

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“…Each sample was then diluted with dephosphorylated PKR and activation buffer to give final conditions of TEN 50 M 5 . In contrast to Figure 2B, monovalent cation concentration was decreased from 100 to 50 mM NaCl: decreasing monovalent salt at early time points has been shown to enhance activation signal (Heinicke et al 2011). The 1/99 WT isolated as dimer remained z75% dimer after native gel purification (Fig.…”

Section: Activation Of Pkr By Purified Hdv Dimermentioning

confidence: 77%

“…Activation of PKR by monomer/dimer mixtures of 1/99 HDV variants Our lab has been interested in identifying and characterizing RNA motifs that regulate PKR activity (Zheng and Bevilacqua 2004;Nallagatla et al 2007;Heinicke et al 2009Heinicke et al , 2011Toroney et al 2010); we thus chose to examine activation of PKR by the semiglobular HDV ribozyme. Various lengths of the HDV genome, including wild-type uncleaved and cleaved, and the catalytically unreactive C75U forms of -54/271, -54/186, -54/172, -54/140, and -54/99 were initially tested for PKR activation.…”

Section: Resultsmentioning

confidence: 99%

“…1B). (Note that we avoided weakening P4 through the introduction of bulges, as this would result in bulges in the concomitant dimers, which would very likely prevent PKR activation as recently shown [Heinicke et al 2011].) For this study, all three 1/99 variants were prepared from self-cleaved transcripts that begin at -54 and end with 99 and were isolated on a denaturing 6% PAGE.…”

Section: Resultsmentioning

confidence: 99%

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Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Heinicke

Bevilacqua

2012

RNA

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Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. We report that the HDV ribozyme sequence alone can activate PKR. To elucidate the RNA structural basis for this, we prepared a number of HDV variants, including those with shortened or lengthened P4 pairing regions, with the anticipation that lengthening the P4 extension would yield a more potent activator since it would offer more base pairs of dsRNA. Surprisingly, the variant with a shortened P4 was the most potent activator. Through native gel mobility and enzymatic structure mapping experiments we implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease.

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Section: Activation Of Pkr By Purified Hdv Dimermentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Heinicke

Bevilacqua

2012

RNA

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“…In particular, complex tertiary structures including pseudoknots [7], IRESs [8], and UTRs [9] activate PKR, as well as a number of misfolded or dimerized functional RNAs [10–12]. Multiple RNA secondary structures have been reported to promote activation of PKR including stem-loops containing defects such as smaller bulges [13]. Moreover, certain unstructured RNA regions promote activation.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA

Hull

Bevilacqua

2015

Journal of Molecular Biology

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The protein kinase PKR is a sensor in innate immunity. PKR autophosphorylates in the presence of dsRNA enabling it to phosphorylate its substrate, eIF2α, halting cellular translation. Classical activators of PKR are long viral dsRNAs, but recently PKR has been found to be activated by bacterial RNA. The features of bacterial RNA that activate PKR are unknown, however. We studied the B. subtilis trp 5’-UTR, which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5’-UTR binds a protein, TRAP, which recognizes L-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/L-Trp-bound forms. Segments from the 5’-UTR, including the terminator, 5’-stem-loop, and Shine-Dalgarno blocking hairpins, demonstrated 5’-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5’-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5 mM, the trp 5’-UTR continued to activate PKR potently. Moreover, total RNA from E. coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome.

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“…These nonhelical regions often have specific structural features that are defined by the sequence and hence, become recognition sites for biomolecules or ions (Peattie et al 1981;Wu and Uhlenbeck 1987;Gott et al 1991;Correll et al 1997;Heinicke et al 2011). A bulge loop forms when one or more nucleotides are present on one strand in a duplex region.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

Strom

Shiskova

Hahm

et al. 2015

RNA

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Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1-to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl 2 or 1 M KCl. The DG W 37 loop parameters for 1-to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔDG W values relative to 1 M KCl were 0.47-2.06 kcal/mol more favorable for the RNA bulge loops. The DG W 37 loop parameters for 1-to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4-and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1-to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1-to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride.

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RNA helical imperfections regulate activation of the protein kinase PKR: Effects of bulge position, size, and geometry

Cited by 18 publications

References 38 publications

Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

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