Abstract
How premature translation termination codons (PTCs) mediate effects on nuclear RNA processing is unclear. Here we show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice caused a decrease in the accumulated levels of doubly spliced R2 relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of the large intron splice sites and by mutation of the AUG that initiated translation of R1 and R2. In contrast to the PTC at nt 385, the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor the normal termination codon for NS2; however, it could be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 open reading frame. This suggested that there was recognition and communication of reading frame between exons on a pre-mRNA in the nucleus prior to or concomitant with splicing.
Highlights
Premature termination codons (PTCs)1 have been shown to result in decreased levels of PTC-containing mRNAs in many organisms, and there is an increasing appreciation of the effect of PTCs on RNA levels in mammalian cells
Because of Effects on Excision of the Downstream Large Intron—Introduction of an ochre (TAA) PTC at nt 385 in the NS1/NS2 common exon of P4-generated pre-mRNA, either by point mutagenesis or by insertion of a 15-nt linker with overlapping PTCs in all three reading frames, led to a significant inversion in the accumulated levels of doubly spliced R2 relative to singly spliced R1 in total RNA, compared with that generated by wild type, the total P4-generated product (R1ϩR2) was unchanged (Fig. 2, A and B)
A PTC transversion mutation but not a missense transversion mutation at nucleotide 385 in the 5Ј-terminal NS1/2 common exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice (MVM) resulted in an increased level of the singly spliced R1 mRNA relative to doubly spliced R2 mRNA in the nucleus of transfected cells
Summary
Comprised of 5Ј and 3Ј elements within the NS2-specific exon [17]. The function of this ESE is sensitive to the presence of PTCs [5]. When the PTC at nt 2018 was combined with a mutation in the 3Ј element of the bipartite enhancer, the NS2-specific exon was skipped These effects were independent of RNA stability and were shown to be reading frame-dependent; single nucleotide deletions in front of the 2018 mutation, which removed the PTC from the NS2 open reading frame, fully suppressed its effect [5]. The effect of the PTC at nt 2018 could, be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 ORF, suggesting that there was communication of reading frames between exons. Our observations are most consistent with a model in which reading frame can be recognized on a pre-mRNA molecule in the nucleus, prior to or concomitant with splicing
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