Structural probing of a pathogenic tRNA dimer

Roy, Marc D.; Wittenhagen, Lisa M.; Kelley, Shana O.

doi:10.1261/rna.7143305

Cited by 30 publications

(39 citation statements)

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“…Number of nucleotides in cleaved monomer HDV is provided above the gel. The faster migrating bands are assigned as monomer (M) and the slower migrating bands as dimer (D) as described in the Materials and Methods; assignment of these bands is facilitated by structure mapping (see below) and by comparison to lanes 1 and 2, which are tRNA monomer (M) and dimer (D), with the dimer prepared as per Wittenhagen and Kelley (2002) and Roy et al (2005). ( a bell-shaped dependence on RNA concentration for the most potent RNA activator (see Fig.…”

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: Resultsmentioning

confidence: 99%

“…2A), prepared as described elsewhere (Wittenhagen and Kelley 2002;Roy et al 2005). The 1/99 D3-bp WT, at 93 nt, is 15 nt or 19% longer than tRNA.…”

Section: Native Gel Analysis Of Hdv Dimerizationmentioning

confidence: 99%

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

Heinicke

Bevilacqua

2012

RNA

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“…21 The dimer disrupts native tertiary structure and approximately doubles the number of base pairs, from 20 to 42 bp (Figure 2C). 32 As described below, covalent RNA modifications occur much less frequently in mitochondrial tRNAs than cytoplasmic tRNAs. Since tRNAs are prone to misfold, modifications in cytoplasmic tRNAs, which are known to favor native tertiary structure, may function, at least in part, to suppress tRNA dimerization thereby preventing an unprovoked innate immune response.…”

Section: Pkr Is Activated By Rna Misfolding and Dimerizationmentioning

confidence: 99%

Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR

Hull

Bevilacqua

2016

Acc. Chem. Res.

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CONSPECTUS Pathogens are recognized by the innate immune system in part via their unique and complex RNA signatures. A key sensor in human innate immunity is the RNA-activated protein kinase PKR, which has two double-stranded RNA (dsRNA) binding motifs (dsRBMs) at its N-terminus. Early studies described PKR as being activated potently by long stretches of perfect dsRNA, a signature typical of viruses. More recently, we and others have found that PKR is also activated by RNAs having structural defects such as bulges and internal loops. This article describes advances in our understanding of the ability of PKR to detect diverse foreign RNAs and how that recognition plays significant roles in discriminating self from non-self. The experiments discussed employ a wide range of techniques including activation assays, native polyacrylamide gel electrophoresis (PAGE), protein footprinting, and small angle X-ray scattering (SAXS). We discuss how misfolding and dimerization of RNA lead to activation of PKR. We also present recent findings on the activation of PKR by varied bacterial functional RNAs including ribozymes and riboswitches, which are among the few structured RNAs known to interact with PKR in a site-specific manner. Molecular models for how these structured RNAs activate PKR are provided. Studies by SAXS revealed that PKR straightens bent RNAs. Most external and internal RNA cellular modifications introduced in vitro and found naturally, such as the m7G cap and m6A group, abrogate activation of PKR, but other modifications, such as 5’-ppp and 2’-fluoro groups, are immunostimulatory and potential anticancer agents. Genome-wide studies of RNA folding in vitro and in vivo have provided fresh insights into general differences in RNA structure amongst bacteria, viruses, and human. These studies suggest that in vivo, cellular human RNAs are less folded than once thought, unwound by helicases, destabilized by m6A modifications, and often bound up with proteins—conditions known to abrogate activation of PKR. It thus appears that non-self RNAs are detected as unmodified, naked RNAs with appreciable secondary and tertiary structure. Observation that PKR is activated by structured but otherwise diverse RNAs is consistent both with the broad-spectrum nature of innate immunity and with the non-specific recognition of RNA by the dsRBM family. These findings provide a possible explanation for the apparent absence of protein-free structured human RNAs, such as ribozymes and riboswitches.

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“…Several studies suggest that tRNAs have a tendency to dimerize and that such dimers are separable by analytical techniques. Examples of dimerizing tRNAs include tRNA Tyr [45] and tRNA Glu [46] from E.coli; tRNA Ala from yeast [47]; and a mutant form of mitochondrial tRNA Leu [48,49]. Given that modifications in tRNA strengthen tertiary structure [41][42][43], one function of modifications might be to favor native RNA tertiary structure and thereby minimize tRNA dimerization.…”

Section: Introductionmentioning

confidence: 99%

Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR

et al. 2013

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Interferon inducible protein kinase PKR is an essential component of innate immunity. It is activated by long stretches of dsRNA and provides the first line of host defense against pathogens by inhibiting translation initiation in the infected cell. Many cellular and viral transcripts contain nucleoside modifications and/or tertiary structure that could affect PKR activation. We have previously demonstrated that a 5′-end triphosphate–a signature of certain viral and bacterial transcripts–confers the ability of relatively unstructured model RNA transcripts to activate PKR to inhibit translation, and that this activation is abrogated by certain modifications present in cellular RNAs. In order to understand the biological implications of native RNA tertiary structure and nucleoside modifications on PKR activation, we study here the heavily modified cellular tRNAs and the unmodified or the lightly modified mitochondrial tRNAs (mt-tRNA). We find that both a T7 transcript of yeast tRNAPhe and natively extracted total bovine liver mt-tRNA activate PKR in vitro, whereas native E. coli, bovine liver, yeast, and wheat tRNAPhe do not, nor do a variety of base- or sugar-modified T7 transcripts. These results are further supported by activation of PKR by a natively folded T7 transcript of tRNAPhe in vivo supporting the importance of tRNA modification in suppressing PKR activation in cells. We also examine PKR activation by a T7 transcript of the A14G pathogenic mutant of mt-tRNALeu, which is known to dimerize, and find that the misfolded dimeric form activates PKR in vitro while the monomeric form does not. Overall, the in vitro and in vivo findings herein indicate that tRNAs have an intrinsic ability to activate PKR and that nucleoside modifications and native RNA tertiary folding may function, at least in part, to suppress such activation, thus serving to distinguish self and non-self tRNA in innate immunity.

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Structural probing of a pathogenic tRNA dimer

Cited by 30 publications

References 50 publications

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

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

Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR

Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR

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