Dynamic refolding of IFN-γ mRNA enables it to function as PKR activator and translation template

Cohen-Chalamish, Smadar; Hasson, Anat; Weinberg, Dahlia; Namer, Lise Sarah; Banai, Yona; Osman, Farhat; Kaempfer, Raymond

doi:10.1038/nchembio.234

Cited by 71 publications

(110 citation statements)

References 31 publications

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“…Activation of translation of negative regulators by IL-1 (Table 1) may achieve the same goal, eventual termination of the response, as IFN-␥-induced inhibition of translation; IFN-␥, which itself is translationally controlled by a PKR-activating sequence spanning part of its 5Ј UTR and coding sequences (72,73), restricts translation of some of its target genes through a heteromeric complex assembled at the target mRNAs (74).…”

Section: Il-1-induced Post-transcriptional Controlmentioning

confidence: 99%

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*

Dhamija

Doerrie

Winzen

et al. 2010

Journal of Biological Chemistry

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The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IB, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1␣-treated HeLa cells. Polysome profiling of IB mRNA and reporter mRNAs carrying its 3 UTR indicated poor translation in unstimulated cells. 3 UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of capindependent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5 sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.Members of the IL-1 family of cytokines are intricately involved in inflammatory and immune responses (1). The founding members IL-1␣ and -␤ exert their multiple activities by binding to the same species of heterodimeric cell surface receptors present on virtually all nucleated cells. Consequences of their activation include changes in expression of numerous genes through activation of NF-B and the JNK and p38 MAPK cascades (2-5). In addition to transcriptional activation, posttranscriptional mechanisms have been reported to contribute to these changes. mRNA stabilization in response to IL-1 as well as LPS and other inflammatory activators depends on the p38 MAPK/MK2 pathway, which affects proteins that control degradation of mRNAs through selective binding to AU-rich elements (AREs) 3 (6, 7). The PI3K and JNK pathways have been reported as well to lead to stabilization of mRNAs (8, 9). In addition, IL-1 induces stabilization of several mRNAs independently of TRAF6/p38/MK2 through an as yet unidentified signaling pathway downstream of IRAK1 (10).The amount of a protein synthesized by the cell in a given condition is finally controlled at the level of translation by mechanisms related to those controlling mRNA degradation (11). Translational control of gene expression can affect most RNAs as an adaptation of general protein synthesis to cell growth stimulation or stress conditions, or it can specifically target a small group of mRNAs (12, 13). This study provides an example f...

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Section: Il-1-induced Post-transcriptional Controlmentioning

confidence: 99%

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*

Dhamija

Doerrie

Winzen

et al. 2010

Journal of Biological Chemistry

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“…These three helical segments together would make a 56-bp dsRNA, which would be a strong PKR activator, as observed herein. The ability of multiple shorter helical fragments to promote activation has been noted previously in aptamers, pseudoknots, TAR, and the HCV IRES (Bevilacqua et al 1998;Cohen-Chalamish et al 2009;Shimoike et al 2009;Heinicke 2010;Toroney et al 2010). We note that similar activation of PKR by dimers of 1/99 D3 bp, WT, and +3 bp constructs at 0.1 mM RNA dimer (Supplemental Fig.…”

Section: Secondary Structure Mapping Of Hdv Monomers and Dimersmentioning

confidence: 70%

“…In addition, PKR is known to be activated by highly structured RNA domains of hepatitis C virus (HCV) (Shimoike et al 2009;Toroney et al 2010) and a pseudoknotted human IFN-g-mRNA (Ben-Asouli et al 2002;Cohen-Chalamish et al 2009). Unlike the above activators, the HDV ribozyme is semiglobular, although it contains a protruding 13-bp pairing region (P4).…”

Section: Introductionmentioning

confidence: 99%

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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“…TPT1 mRNA activates the double-stranded RNA (dsRNA) recognition protein PKR (Bommer et al 2002;Nussbaum et al 2002). PKR primarily Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by rnajournal.cshlp.org Downloaded from recognizes viral dsRNA and inhibits viral translation, but PKR also interacts with a variety of cellular dsRNA-containing mRNAs, including the TNF-α and IFN-γ (Osman et al 1999;Cohen-Chalamish et al 2009;Hull and Bevilacqua 2016). Activation of PKR depends on a dsRNA region of at least 30 bases to allow binding of two or more PKR molecules (Manche et al 1992;Zheng and Bevilacqua 2004;Lemaire et al 2008).…”

Section: Lackey 12mentioning

confidence: 99%

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

Lackey

Coria

Woods

et al. 2018

RNA

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The precise impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next generation sequencing to obtain experimental structure data, such as SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension and Mutational Profiling), can reveal structural effects of mutations especially when this data is rigorously incorporated in RNA structural modeling. Here we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single nucleotide mutations. We find that allele-specific sorting greatly improved the detection ability of SHAPE-MaP. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of non-coding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1. Combined with rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble we identified a subset of somatic and Lackey 2 inherited mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified acting as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures are on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both TPT1 and LCP1 have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro are likely to exist under biological conditions. Overall, these data reveal a remarkably robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with a benchmark for further algorithmic development.

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Dynamic refolding of IFN-γ mRNA enables it to function as PKR activator and translation template

Cited by 71 publications

References 31 publications

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*

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

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

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