An Ancient Pseudoknot in TNF-α Pre-mRNA Activates PKR, Inducing eIF2α Phosphorylation that Potently Enhances Splicing

Abstract

Tumor necrosis factor alpha (TNF-α) is expressed promptly during inflammatory responses. Efficient TNF-α mRNA splicing is achieved through a 3' UTR element that activates RNA-dependent eIF2α protein kinase (PKR). The TNF-α RNA activator, we show, folds into a pseudoknot conserved from teleost fish to humans, critical for PKR activation and mRNA splicing. The pseudoknot constrains the RNA into two double-helical stacks having parallel axes, permitting facile PKR dimerization and trans-autophosphorylation needed for kinase activation. Mutations show that the PKR activator potently enhances splicing without inhibiting translation. eIF2α phosphorylation represses translation and is essential for coping with cellular stress, yet PKR-enabled TNF mRNA splicing depends strictly on eIF2α phosphorylation. Indeed, eIF2α phosphorylation at Serine51 is necessary and sufficient to achieve highly efficient splicing, extending its role from negative control of translation to positive control of splicing. This mechanism, operational in human peripheral blood mononuclear cells (PBMCs), links stress signaling to protective immunity through TNF mRNA splicing rendered efficient upon eIF2α phosphorylation.

Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

“…It is possible, however, that such higher-order interactions, which are difficult to identify, are common in eukaryotic translation initiation. Indeed, pseudoknots constitute a well-known structural motif 87 in bacterial riboswitches and ribozymes and have roles in eukaryotic pre-mRNA processing such as in splicing 88,89 and adenosine-to-inosine editing 90 . Furthermore, there are prominent examples of cis- regulatory pseudoknots in the CDS that interact directly with translating ribosomes to induce programmed frameshifting (reviewed in REFS 91–93).…”

CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts

“…2c ). To further demonstrate that G 4 C 2 RAN translation starts with a methionine, we inhibited the activity of the methionylated initiator tRNA Met carrier eIF2 by inducing the phosphorylation of its α subunit with poly(I:C)/salubrinal treatment as previously described 41 (Supplementary Fig. 4a ).…”

The search for a PKR code—differential regulation of protein kinase R activity by diverse RNA and protein regulators

“…2). In this model, binding of one PKR monomer to each parallel helix could bring them into close proximity and allow for on-RNA dimerization and subsequent activation (Namer et al 2017). Surprisingly, no inhibition of translation, including that of mature TNF-α mRNA, following PKR activation by 2-APRE was observed.…”

“…Tumor necrosis factor-α (TNF-α) is a cytokine regulated by splicing and is essential in protective immunity and inflammatory response. A 2-aminopurine response element (2-APRE), located in the TNF-α3 ′ -UTR, binds and activates PKR in vivo and in vitro (Namer et al 2017). A conserved pseudoknot that aligns two parallel coaxially stacked helices, individually long enough to accommodate one PKR monomer, was proposed to drive PKR activation (Fig.…”

“…Surprisingly, no inhibition of translation, including that of mature TNF-α mRNA, following PKR activation by 2-APRE was observed. Importantly, the splicing of TNF-α pre-mRNA relied on PKR autophosphorylation and downstream phosphorylation of eIF2α (Namer et al 2017). This novel splicing-based mechanism may provide a fast and efficient way to respond to viral infection or stress.…”