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. 2017 Apr 4;27(5):688–704. doi: 10.1038/cr.2017.39

Figure 5.

Figure 5

Splicing efficiency is coupled to activation of PKR by the activator in β-globin exon 1. (A) Activation of PKR by β-globin RNA. Phosphorylation of rPKR was assayed in the presence of [γ-32P]ATP, and 5 and 10 nM intact β-globin pre-mRNA template (nts 1-477) or truncated RNAs (nts 1-124, 5-124 and 1-118). (B) In-line structure probing of the 5′-terminal 124 nts of the β-globin pre-mRNA template. Gel shows representative pattern of spontaneous cleavages in 5′ end-labeled RNA incubated alone (−) or with rPKR (nM) as shown. G (T1) and alkali (OH) ladders serve as size markers; NR, input RNA. Structure on left is based on repeated in-line probing. Red and green colors denote nt showing enhanced and reduced cleavage, respectively, induced by rPKR. In helix S1, boxes denote nts mutated in s1a and s1b. (C-E) In s1a, positions 47-51 were mutated to GUGGU; in s1b, positions 110-114 were mutated to GUACC (red sequences in C). Activation of rabbit reticulocyte PKR (C, E) or rPKR (D) by wt or mutant forms of the 477-nt (C, D) or 124-nt (E) RNA was assayed; eIF2α phosphorylation is shown in E. For activation of rabbit reticulocyte PKR, 1.2 and 2 nM RNA was used, and for rPKR, 5 and 10 nM RNA was used. (F, G) Splicing efficiency is coupled to the ability to activate PKR. (F) Splicing of wt and mutant forms of β-globin pre-mRNA template in HeLa cell nuclear extract was analyzed as in Figure 1F; left four lanes, input unspliced RNA (U). (G) Comparison of the ability to activate PKR at the lower RNA concentration in D and splicing efficiency (ratio of spliced RNA over pre-mRNA) in F. (H) Splicing of s1b pre-mRNA template is restored by compensatory mutation. In vitro splicing was assayed in the absence or presence of αPeIF2α (1 μg); splicing efficiency is plotted in the bar graph. (I) β-globin precursor RNA processing is coupled to the ability to activate PKR. HEK293T cells were transfected with wt- or s1b-mutant β-globin gene and excision of intron 1 was assayed by quantitative RT-PCR; ratio of unspliced over spliced RNA is plotted (means + SEM; n = 3).