Nonsense-mediated mRNA decay (NMD) selectively recognizes and targets for degradation mRNAs containing premature termination codons. This mRNA quality control mechanism prevents potentially deleterious dominant negative effects of truncated proteins that accumulate if aberrant mRNAs are not degraded [1–4]. In mammalian cells, NMD proteins can efficiently identify a termination codon as premature if the stop codon resides at least 50 nucleotides upstream of the terminal exon-exon junction [5, 6].
When introns are removed during splicing, a multi-protein complex called the exon junction complex (EJC) is deposited on the mRNA 20-24 nucleotides upstream of the exon-exon junction . When a translating ribosome encounters a termination codon, it pauses; and the eukaryotic release factors, eRF1 and eRF3, as well as the NMD factors Upf1 and Smg1, are recruited . If the termination codon is premature, Upf1 will interact with the downstream EJC via two additional NMD factors, Upf2 and Upf3b. This forms a decay-inducing complex that signals a premature termination event . The mRNA is then rapidly targeted for degradation in the cytoplasm so that it is no longer translated. In most mRNA transcripts, the natural termination codon resides in the final exon of a spliced transcript, preventing the occurrence of a downstream EJC .
NMD poses a unique risk to the genome and mRNAs of retroviruses. Although retroviruses encode some enzymatic activities, they rely on the host cell's reservoir of proteins to produce progeny virions. As a result of this dependence on host cell machinery, retroviruses must overcome mRNA quality control measures to ensure their genome is translated in an efficient and timely manner. The genomes of simple retroviruses, such as the Rous sarcoma virus (RSV), possess cis-acting RNA elements that play an essential role in facilitating successful genomic expression [10–13].
During the RSV life cycle, expression of the integrated proviral DNA generates three viral mRNAs that are capped and polyadenylated: two spliced and one unspliced [14, 15]. Full-length, unspliced 9.3 kb viral RNA is exported to the cytoplasm where it not only becomes the genome of progeny virions, but also acts as the mRNA template for Gag and Gag-Pol polyproteins . This viral mRNA is presented to the host translation machinery with characteristics rarely observed among host cell mRNAs: a long 3' UTR, retained introns, and multiple open reading frames. As a result of these mRNA features, the full-length viral RNA should be recognized by the host NMD machinery and degraded; however, the RNA is stable with a half-life of ~7-20 hours [17, 18].
Premature termination codons within the open reading frame of gag result in a decrease in unspliced viral RNA levels . This decay relies upon the central NMD protein Upf1 and translation of the viral RNA, thereby implicating the NMD machinery in differentiating premature from natural termination codons in this unspliced viral RNA . Thus, full-length viral RNA is not immune to host mRNA decay surveillance as has been observed for some intronless mRNAs in mammalian cells [21, 22]. The gag open reading frame of RSV is removed from all spliced viral mRNAs; therefore a model that relies upon downstream exon junction complexes for recognition of a premature termination codon is unsatisfactory in the context of the RSV viral RNA. In fact, recent studies have suggested that an EJC is not required for recognition by NMD [22, 23].
An alternative model in vertebrates proposes that NMD is induced when the termination codon is distant from the polyA tail and the polyA binding proteins [22–24]. The distance between the natural stop codon and the polyA tail is usually relatively short. In humans 80% of polyA tails are within 2 kb of the translation termination codon . When a premature termination codon arises within the open reading frame, it would be a greater distance from the 3' polyA tail. In support of this model, some transcripts with long 3' UTRs are unstable and degraded by NMD [22, 23, 26–28]. The unspliced viral RNA is polycistronic, but Gag is the major protein product generated from this mRNA resulting in an apparent 3' UTR of over 7 kb. The average length of a 3' UTR in chicken cells is approximately 600 nucleotides, with over 80% of the polyA tails being within 1200 nucleotides of the translation termination codon [29, 30]. Again, a model where the distance from a stop codon to the polyA tail would determine whether a termination codon is premature is difficult to reconcile in the context of RSV. Therefore, we propose that an alternative mechanism must exist to allow the NMD machinery to identify premature termination codons within RSV RNA.
During initial efforts to characterize the decay of unspliced RSV RNA, it was noted that deletions downstream of gag decreased unspliced viral RNA levels . When 400 nucleotides downstream of gag are deleted or inverted, unspliced viral RNA levels are reduced to quantities comparable to viral constructs containing a premature termination codon within gag . This cis RNA element was termed the Rous sarcoma virus stability element (RSE). Furthermore, when the RSE is inserted after a premature termination codon within the gag open reading frame, the viral RNA no longer undergoes decay . This suggests that the RSE generates a signal to identify the correct termination codon.
We sought to define key RNA features of the RSE through directed mutagenesis of the virus. In this report we describe RNA sequence features that play a role in RSE function. These data suggest that the RSE is comprised of structure and sequence components with many redundant sub-elements. These elements function independently of the nucleotide sequence of the termination codon and the first nucleotide following the termination codon. Furthermore, the 3'UTRs of the other RSV open reading frames of the parental avian leukosis virus (ALV) may also function as stability elements.