A mutagenesis approach showed that the two Dot1 isoforms are produced from two alternative translation start sites as a result of leaky scanning by the ribosome. the resistance of yeast cells to the chitin-binding drug Calcofluor White, suggesting that the two Dot1 isoforms have a differential function in cell wall biogenesis. == INTRODUCTION == Dot1 is a histone methyltransferase that was originally identified inSaccharomyces cerevisiae. The enzyme, which is conserved from yeast to humans, catalyzes mono-, di- and trimethylation DL-alpha-Tocopherol methoxypolyethylene glycol succinate of lysine 79 on histone H3 (H3K79), a residue located on the nucleosome core (14). In yeast, Dot1 methylates 90% of histone H3 and is involved in gene silencing, activation of the DNA damage response and the pachytene checkpoint in meiosis (1,57). In addition, genetic interactions have been reported betweenDOT1and different genes involved in cell wall biogenesis, suggesting a function for Dot1 in this process (8,9). In mammals, Dot1 plays a role in Ras-induced gene silencing and aldosterone-induced gene repression as well as in inappropriate gene activation in certain types of leukemia (1013). Our antibodies directed against yeast Dot1 detect two protein species on immunoblots, even though the protein is encoded by a single gene (1). Several mechanisms can result in the formation of two DL-alpha-Tocopherol methoxypolyethylene glycol succinate different protein species from one gene. First, full-length proteins can be processed by proteases, resulting in the generation of N- or C-terminally truncated forms. Second, several posttranslational modifications such as phosphorylation or ubiquitination can result in an altered mobility of proteins on immunoblots. Finally, different protein isoforms can be generated by alternative translation, which can be caused by variations in the mRNA sequence (for example due to alternative mRNA splicing or alternative transcription start site selection) or by the usage of multiple translation start sites. Translation from multiple start codons can occur when the mRNA contains an internal ribosomal entry site (IRES), or when the ribosome scanning over the mRNA molecule skips the first start codon and subsequently initiates translation from a downstream alternative start codon, a phenomenon called leaky scanning (14). Alternatively, the ribosome can bind to the mRNA on the 5-side of the start codon via the normal mechanism, but then jumps over the first start codon to initiate transcription from a downstream start codon, a process called Rabbit Polyclonal to HEY2 ribosome shunting (15). Translation start site selection is a highly regulated process that is critical for differential gene regulation in eukaryotes (16). Mutations that affect translation initiation have been associated with a range of genetic diseases in humans (17). However, there are still many open questions regarding the molecular mechanisms of start DL-alpha-Tocopherol methoxypolyethylene glycol succinate codon selection (18). In this study, we determined the mechanism by which the two Dot1 isoforms are generated. We show by protein tagging, protein truncation and mutational analysis that they are the result of leaky scanning by the ribosome. This enabled the construction of yeast strains expressing either one of the isoforms. The two Dot1 isoforms were found to have DL-alpha-Tocopherol methoxypolyethylene glycol succinate indistinguishable functions in global methylation and gene silencing. However, they showed distinct functions in resistance to the chitin-binding molecule Calcofluor White (CFW), suggesting that they play different roles in cell wall biogenesis. Our results suggest that leaky scanning of theDOT1mRNA is affected by sequences downstream of the start codon, whereas the efficiency of translation initiation DL-alpha-Tocopherol methoxypolyethylene glycol succinate is usually thought to depend mainly on the 5-context of the start codon (14). The discovery of unconventional leaky scanning events like the one reported here will be instrumental to unravel the complex mechanisms that regulate translation initiation in yeast and higher eukaryotes. == MATERIALS AND METHODS == == Yeast strains, plasmids and media == Yeast strains and plasmids used in this study are listed inTable 1. Yeast media were described previously (1). Silencing assays were performed using media containing 1 g/l.