Positive Regulation of Splicing of Cellular and Viral mRNA by Intragenic RNA Elements That Activate the Stress Kinase PKR, an Antiviral Mechanism

Figure 1. Regulation of splicing of TNF-α pre-mRNA by an intragenic element that induces activation of PKR, resulting in eIF2α phosphorylation. Exons are in dark blue. The PKR activator element of 104 nucleotides in length (2-APRE) that maps into the TNF-α 3′-UTR (red) strongly activates PKR within the nucleus [13]. This RNA element folds into a pseudoknot structure that causes the RNA to be constrained into two parallel helices, each capable of binding to a single PKR monomer, thereby facilitating the formation of PKR dimer on the 2-APRE RNA that is needed for the activation of the kinase [10]. The active PKR dimer next must induce the phosphorylation of nuclear eIF2α in order to enable the highly efficient excision of the introns (yellow) from TNF-α pre-mRNA [10]. The mature TNF-α mRNA splicing product is exported from the nucleus into the cytoplasm and is translated without impediment [10,13,19].

Figure 2. Control of splicing of β-globin pre-mRNA by an intragenic RNA element that activates PKR and silencing of the PKR activator upon excision of the first intron. Within the human β-globin RNA element that activates PKR, which consists of the 5′-terminal 124 nt of exon 1, the 5-bp helix a-b (green) plays a critical role in PKR activation. Once PKR has been activated, it must phosphorylate its eIF-2α substrate in order to promote the assembly of early spliceosomes and allow for the efficient splicing of β-globin mRNA. Once intron 1 is excised, sequence c, that is located close to the beginning of exon 2 (red) base-pairs with strand a and thereby displaces strand b, inducing a structural rearrangement within the RNA that results in silencing of the ability of the mature spliced β-globin mRNA to activate PKR. This structural rearrangement causes the activation of PKR to be transitory, serving only to enhance splicing while avoiding the inhibition of β-globin protein synthesis that is essential for survival [14,19].

Figure 3. Splicing of HIV rev/tat mRNA is dependent on the activation of PKR by an intragenic pseudoknot located just upstream of 3′TAR and by the 3′TAR stem-loop, inducing eIF2α phosphorylation [18]. Local activation of PKR within the cell, predominantly by the pseudoknot element (PK) yet also by 3′TAR (1), induces eIF2α phosphorylation (2) that triggers excision of the large rev/tat intron (3). Mutations (boxed) within pseudoknot stem P1 or within the stem of 3′TAR each cause a severe impairment in splicing of rev/tat mRNA [18].