A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Abstract

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5 element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5 and 3 elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3 enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3 splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.Premature termination codons (PTCs) decrease the accumulated levels of most known mRNAs in which they reside (reviewed in reference 28). In the yeast Saccharomyces cerevisiae, the UPF genes are involved in the degradation of PTCcontaining RNAs in the cytoplasm (review in reference 37). Similarly, the SMG proteins of Caenorhabditis elegans are required for the rapid decay of PTC-containing unc-54 myosin heavy-chain mRNAs (reviewed in reference 41).PTC-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases (reviewed in references 28 and 29); however, how PTCs mediate such effects in the nucleus is still unclear. In mammalian cells, PTCs have been shown to decrease the levels of nucleus-associated mRNAs by a posttranscriptional mechanism, often attributed to mRNA decay (3,6,9,28,45). Experiments done with PTCs in the human TPI gene suggest that nonsense-mediated decay (NMD) occurs on a fully spliced nucleus-associated mRNA molecule, possibly during mRNA export to the cytoplasm (4, 5, 9, 28).There is also increasing evidence that PTCs may affect mammalian RNA processing events other than decay. For example, several instances of PTC-associated exon skipping have been described (15,19,28,33). Perhaps the best-studied example is skipping of the 66-nucleotide (nt) exon 51 of fibrillin FBN1 RNA, which was detected when nonsense but not missense mutations were present within this exon, which was independent of protein synthesis, and for which normal splicing was restored when the nonsense codon was shifted out of frame with the initiation codon (14, 23). Retention of introns upstream of PTC-containing exons has also been reported for P4-ge...

A Premature Termination Codon Mutation at the C Terminus of Foamy Virus Gag Downregulates the Levels of Spliced pol mRNA

“…Nonsense codons acquired during normal lymphocyte development have been shown to accumulate incompletely spliced or unspliced pre-mRNA for several genes in the nucleus (immunoglobulin and and T-cell receptor ␤) (16,23). It has also been shown that PTC, but not missense mutations, inserted into the genome of the parvovirus minute virus of mice (MVM) inhibited nuclear excision of an intron upstream of a PTC-containing exon, leading to the retention of the upstream intron (9). This is somewhat similar to the FV case, since our PTC mutations are in the exon downstream of the pol 3Јss and MVM also has a poor polypyrimidine tract in the 3Јss.…”

Polypyrimidine Track-binding Protein Binding Downstream of Caspase-2 Alternative Exon 9 Represses Its Inclusion

“…In a few instances, two different types of splicing enhancers (e.g. Pu-rich and CA-rich) were found in the same exon (51)(52)(53)(54). Another example of an intronic repressor that can be separated into two mechanistically distinct subelements was identified in the hnRNP A1 pre-mRNA downstream of the alternative exon 7B; however, the mechanism of action is different (55).…”

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

“…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.…”