Academic journal article Genetics

Nonsense-Mediated Decay of Ash1 Nonsense Transcripts in Saccharomyces Cerevisiae

Academic journal article Genetics

Nonsense-Mediated Decay of Ash1 Nonsense Transcripts in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.

IN eukaryotes, nonsense-mediated mRNA decay (NMD) plays a role in RNA surveillance by eliminating aberrant transcripts that contain a nonsense or frameshift mutation, thereby preventing the accumulation of potentially deleterious dominant-negative proteins. In addition, a subset of functional, error-free mRNAs accumulate in a manner dependent on the NMD pathway in the yeast Saccharomyces cerevisiae (Guan et al. 2006), including transcripts with a small upstream open reading frame that initiates translation in the 59- untranslated region (UTR) (Oliveira and McCarthy 1995), transcripts in which aninternal out-of-frame open reading frame (ORF) is translated due to inefficient translation initiation at the firstAUGcodon(Welchand Jacobson 1999), and precursors that undergo ineffi- cient splicing in which the intron contains an in-frame stop codon (He et al. 1993).

The UPF1, UPF2, and UPF3 genes are required for NMD in S. cerevisiae (Leeds et al. 1992). The similarities of UPF gene orthologs from different classes of organisms coincide with similarities in the pathways for NMD, including a recruitment step initiated in the nucleus involving the nucleo-cytoplasmic shuttling proteinUpf3p (Shirley et al. 1998, 2002; Serin et al. 2001), followed by translation initiation, premature termination, decapping, and decay in the cytoplasm. Although NMD can trigger RNA decay during any round of translation in yeast (Maderazo et al. 2003), decay is known to occur during the pioneer round of translation while RNAs are still bound to the nuclear cap-binding complex (Gao et al. 2005).

During pioneer translation, NMD appears to be temporally and spatially coupled to nuclear export. However, in S. cerevisiae, >25 transcripts have been identified where nuclear export and translation are separated by an intervening step in which the transcripts localize via translocation on actin cables. During transport, translation is repressed. Upon arrival and anchoring at the bud tip, translational repression is relieved (Long et al. 1997; Takizawa et al. 2000; Shepard et al. 2003; Andoh et al. 2006; Aronov et al. 2007). ASH1 translation appears to utilize specialized ribosomes containing a specific subset of paralogous ribosomal proteins (Komili et al. 2007; Warner 2007). These exceptional transcripts can be exploited to learn more about NMD.

ASH1 mRNA, the best-studied transcript that localizes via actin cables, codes for a transcriptional repressor of the HO gene, which produces the endonuclease that initiates homothallic switching between a- and a-mating types (Kruse et al. 2002;Gonsalvez et al. 2005; Zarnack and Feldbru?gge 2007). Asymmetric localization of the ASH1 transcript prior to translation leads to asymmetric competence to switch mating type (Chartrand et al. …

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