Figure 4. Northern blot analysis of splicing products.

(A) Twintron scheme with the positions of the four probes used and sizes of likely or expected splicing products; not to scale. The approximated sizes include the estimated 3′ UTR length. (B) High molecular weight blots (1000–6000 nt) for all probes. The three intron probes were used on membranes generated from the same gel and are shown in the original order including size markers. All three probes show similar patterns, the respective linear introns are indicated by arrows. The two probes for the host intron show an identical pattern, but with different relative signal intensities. The apparent band pattern around 1500 nt is an artifact generated by the abundant rRNA bands blocking hybridization. The probe for the mRNA 5′-exon (different gel) detected mainly the mature mRNA (main signal) together with some small intermediates, here the most slowly migrating band is likely the intermediate precursor of ~3,030 nt, which was also detected with both host intron probes. (C) Low molecular weight, high-resolution blots (300–1000 nt) with the same probes. The first, third, and fourth panels show blots originating from the same 5% PAA gel, whereas the second panel was from a different 6% PAA gel, hence the additional marker shown next to it. The probe for the 1^st part of the host intron detected a prominent RNA at ~430 nt, which was also detectable with the 5′-exon probe (black arrows) and represents the free 5′exon generated by SER for inner intron splicing. The main signal detected with the 5′-exon probe was again the mature mRNA. The size of ~370 nt lead to the estimation of a ~60 nt 3′UTR.