Yeast Upf proteins required for RNA surveillance affect global expression of the yeast transcriptome.

Nonsense and frameshift mutations cause premature termination of translation. In conjunction with this, they also trigger nonsense-mediated mRNA decay (NMD), which greatly accelerates the rate of degradation of the mRNA. By decreasing the half-life of the mRNA, nonsense mRNA accumulation is severely limited (15, 16). The phenomenon whereby mRNAs that would otherwise code for potentially deleterious protein fragments are degraded is called RNA surveillance (7, 26). Surveillance occurs in fungi (21), plants (29), nematodes (26), and vertebrates (22). In Saccharomyces cerevisiae, three genes, UPF1, UPF2, and UPF3, are required for NMD (6, 11, 14–16). Sequence homologs of UPF1, an RNA helicase (8, 30), have been identified in Schizosaccharomyces pombe (4), Caenorhabditis elegans (26), Mus musculus (25), and Homo sapiens (1, 25). Comparison of the Upf1p-like proteins shows that they are related by a common central region containing conserved cysteine-rich and ATP-helicase domains flanked by divergent sequences at both ends. These results suggest that NMD pathways in eukaryotic organisms utilize at least one protein in common. This provides a measure of confidence that further studies of NMD in yeast will shed light on NMD in humans as well. The biological purpose of RNA surveillance is to limit the accumulation of aberrant proteins that arise through errors in gene expression. Inefficient splicing of introns is one of the most frequent natural source of errors in gene expression, leading to the production of nonsense mRNAs that code for aberrant proteins. Expression of the CYH2 gene, which codes for a ribosomal protein in S. cerevisiae, is a good example where the consequences of inefficient splicing have been examined (12). The intron in CYH2 pre-mRNA, which contains stop codons, is inefficiently spliced. In wild-type cells, unspliced pre-mRNA is exported (19), translated up to the premature stop codon, and then rapidly degraded by the NMD pathway. When the NMD pathway is inactivated by a null mutation in any of the three UPF genes, the CYH2 pre-mRNA fails to be rapidly degraded and accumulates to a much higher level. The rapid decay of the pre-mRNA prevents the accumulation of a truncated protein that might assemble with ribosomal subunits and impair function. Evidence that the Upf proteins in S. cerevisiae may serve a second purpose in addition to surveillance for errors in gene expression has been mounting. Several naturally occurring, intronless mRNAs whose normal level of accumulation depends on the presence of functional UPF genes have been identified. The accumulation of mRNAs coding for the transcriptional activator Ppr1p and several downstream target genes in the uracil biosynthetic pathway have been reported to be sensitive to inactivation of UPF1 (15, 24). Also, the mRNA encoding Ctf13p, a subunit of the kinetochore, depends on the presence of functional UPF genes (9). The mechanism through which the abundance of naturally occurring mRNAs are controlled by the Upf proteins is not clear. Prior to this study, it was not known how many naturally occurring mRNAs might be affected by loss of UPF function. To assess the global effects of Upf proteins on gene expression, high-density oligonucleotide arrays (HDOA) representing over 6,000 open reading frames (ORFs) in S. cerevisiae were screened for their effects on mRNA accumulation with UPF1, UPF2, and UPF3 were individually inactivated or when all three genes were simultaneously inactivated. Our results indicate that the level of accumulation of hundreds of mRNAs is dependent on the presence of functional UPF genes.