Initiation of Translation Can Occur Only in a Restricted Region of the CYC1 mRNA of Saccharomyces cerevisiae

Abstract

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a ''sensitive'' region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions ؊24 to ؉37 but not in the remaining 3 distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5 cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.Mutationally-altered forms of the CYC1 gene, which encodes iso-1-cytochrome c in the yeast Saccharomyces cerevisiae have been used to elucidate the major features of translation. The deficiencies of iso-1-cytochrome c in certain cyc1 mutants and the corresponding deduced DNA sequences of cyc1 mutations indicated that AUG was the only codon capable of initiating translation at appreciable levels (48, 49), a finding confirmed in yeast cells with other systems (7, 9). Also, early studies indicated that translation of CYC1 mRNA initiated at the most upstream AUG codon (45-47) and that translation could initiate at a relocated AUG initiator codon within a 37-nucleotide region around the normal initiation site (45-49). These results and the results with deletion mutants (2) suggested the lack of any additional sequence requirements for initiation of translation. However, nucleotides preceding the AUG initiator codon at positions Ϫ1 and Ϫ3 slightly influenced initiation of translation, and the introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpi...

Nonsense-mediated mRNA decay of collagen – emerging complexity in RNA surveillance mechanisms

“…These data suggest that neither Pabp or a poly(A) tail are required for NMD in yeast. The faux 39UTR model is also challenged by experiments showing that PTCs close to the initiation codon at the 59 end of CYC1 mRNA elicit strong NMD (Yun and Sherman, 1995). Eukaryotic mRNA transcripts adopt a closed loop conformation via the interaction of eukaryotic translation initiation factor 4 gamma 3 (eIF4G3) at the 59 end with Pabp at the 39 end (Amrani et al, 2008;Imataka et al, 1998;Le et al, 1997;Tarun and Sachs, 1996;Tarun et al, 1997;Wells et al, 1998).…”

“…In this conformation, stop codons close to the translation initiation codon would not be recognized as premature because eRF3 on a ribosome terminating translation would still be able to interact with Pabp ( Fig. 1A) (Hoshino et al, 1999;Silva et al, 2008;Silva and Ramao, 2009;Yun and Sherman, 1995).…”

Effects of Nonsense Mutations on Nuclear and Cytoplasmic Adenine Phosphoribosyltransferase RNA

“…This phenomenon in yeasts has been termed nonsense-mediated mRNA decay, because it is clear that the stability of mature mRNA is affected (28,35). Extensive sitedirected mutagenesis studies of yeasts have identified sequences within the mRNA that are necessary to trigger accelerated mRNA decay (28,48,63,64). The isolation of mutants resistant to general nonsense-mediated mRNA reduction has led to the identification of several genes required for this phenomenon: upf genes in Saccharomyces cerevisiae (16,35) and smg genes in Caenorhabditis elegans (50).…”

Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

“…As alluded to previously, it is well documented that a mRNA can be rendered unstable by the presence of a nonsense mutation in the ®rst two-thirds of a gene, typically by uncovering a sequence which is viewed as an instability element when untranslated (Lim et al, 1992;Belgrader et al, 1993;Hagan et al, 1995;Yun and Sherman, 1995;Zhang et al, 1995). We currently are using an expression system to determine whether a nonsense mutation is present, and we are continuing e orts to sequence additional exons and surrounding segments from the M059J and M059K cell lines.…”

“…A mRNA can be rendered unstable by the presence of a nonsense mutation, normally in the ®rst two-thirds of a gene (Lim et al, 1992;Belgrader et al, 1993;Hagan et al, 1995;Yun and Sherman, 1995;. We have, therefore, undertaken to sequence the remainder of the DNA-PK cs cDNA derived from M059J and K cells.…”