A Premature Termination Codon in Either Exon of Minute Virus of Mice P4 Promoter-generated Pre-mRNA Can Inhibit Nuclear Splicing of the Intervening Intron in an Open Reading Frame-dependent Manner

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. 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. In some of these cases, PTCs have been shown to affect nuclear-associated mRNA abundance by a process termed nonsense-mediated decay, which has been suggested to degrade fully spliced mRNAs in the nucleus, possibly during mRNA export (reviewed in Refs.1 and 2).PTCs have also been implicated in altering nuclear RNA processing events other than decay in mammalian cells, which results in both intron retention and exon skipping, suggesting that PTCs may influence splice site selection (1). In two cases, exon skipping of the 66-nucleotide exon 51 of fibrillin FBN1 RNA (3) and exon skipping and intron retention for the P4-generated pre-mRNAs that generate the nonstructural proteins of the autonomous parvovirus minute virus of mice (MVM) (4, 5), the effects of PTCs on RNA processing have been shown to be reading frame-dependent.MVM is an autonomous parvovirus that is organized into two overlapping transcription units that produce three major classes of RNA (6 -8) (see Fig. 1). Transcripts R1 and R2 are generated form a promoter (P4) at map unit 4 and encode the viral nonstructural proteins NS1 and NS2, respectively, whereas the R3 transcripts are generated from a promoter at map unit 38 (P38) and encode the viral capsid proteins (6, 9). Both NS1 and NS2 play essential roles in viral replication and cytotoxicity (10), and so maintenance of their relative steady state levels, which is controlled at least in part by alternative splicing, is critical to the viral life cycle (Refs. 11-13; reviewed in Ref. 14). All MVM mRNAs generated during infection or following transfection are very stable (15), and no viral proteins are known to p...

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM)

“…One of the mutants expressed no stable NS2 due to a mutation at residue 86 (NS2-am, NS2null) that effectively prevents expression and/or accumulation of the truncated product (Cotmore et al, 1997; Gersappe et al, 1999; Naeger et al, 1992; Ruiz et al, 2006), while the other expressed approximately one sixth of the total wild-type NS2 level (NS2low). Since NS1 expression cannot be detected until 6 hours post-release in cells infected with NS2null viruses (Ruiz et al, 2006), cells were fixed at 6, 12 and 24 hours after release from aphidicolin, stained for NS1, and blind-scored according to the classes identified in Fig 1A.…”

Stop codons affect 5′ splice site selection by surveillance of splicing

“…In the fibrillin gene, associated with Marfan syndrome, nonsense, but not missense, mutations were associated with skipping of exon 51 (29). These studies also include cases of intron retention (30)(31)(32)(33)(34). In particular, nonsense mutations in the Ig gene cause defective splicing of this pre-mRNA in a B cell in vitro system (32).…”

Nuclear translation: What is the evidence?

“…Thus, the efficient return of this factor to the nucleus would depend on translation of the entire coding region of the mRNA, an event that would not occur if the ribosome encountered a PTC during the pioneering round of translation. Comparable sequestration of other sequencespecific splicing factors might account for other forms of splice site selection that lead to skipping of specific PTCcontaining sequences (Gersappe et al 1999;Li et al 2002). Association of the VDJ exon binding protein(s) with cytoplasmic ribosomes might also account for the special, highly efficient NMD (VDJ-exon dependent "Super NMD") to which this TCR-␤ mRNA is subject (Gudikote and Wilkinson 2002).…”