Effective Translation of the Second Cistron in Two Drosophila Dicistronic Transcripts Is Determined by the Absence of In-frame AUG Codons in the First Cistron

Adam A Wall,A Marie Phillips,Leonard E Kelly

doi:10.1074/jbc.m500255200

Adam A Wall, A Marie Phillips + Show 1 more

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https://doi.org/10.1074/jbc.m500255200

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Abstract

The novel dicistronic transcript encoded by the Drosophila melanogaster stoned gene was recognized as being unusual in that the protein encoded by the first open reading frame, stoned-A (STNA), contains no internal methionine residues in a protein of 93 kDa. The dicistronic nature of the stoned locus and the lack of methionine residues in STNA is conserved across dipteran species. A second methionine-free cistron, encoding Snapin, was identified in Drosophila and also found to be dicistronic, the second open reading frame (ORF) encoding a methyltransferase. We have replaced the methyltransferase cistron with green fluorescent protein (GFP) and used this dicistronic construct to show that the GFP cistron is translated in Drosophila S2 cells. The insertion of in-frame AUG codons into the snapin ORF attenuates the translation of GFP, and the level of attenuation correlates with the number of inserted AUGs. Increasing the efficiency of translation-initiation of the Snapin cistron also attenuates the translation of GFP. This indicates that failure to initiate translation at the first AUG allows ribosomes to scan through the Snapin ORF and to initiate translation of the second cistron, unless new AUG codons are inserted. These data are used to interpret the expression of the stoned locus and in particular, to explain the altered stoned protein levels in the stoned-temperature-sensitive mutant allele, which replaces a lysine with a methionine codon early in the first, stonedA, cistron.

Highlights

Identified [4], and a non-viral dicistronic mRNA, with proposed internal ribosome entry sites (IRES) between the cistrons, has been recognized as an alternative transcript from the Adh locus in Drosophila [5]
The novel dicistronic transcript encoded by the Drosophila melanogaster stoned gene was recognized as being unusual in that the protein encoded by the first open reading frame, stoned-A (STNA), contains no internal methionine residues in a protein of 93 kDa
A third possible mechanism for translation of the second open reading frame (ORF) in dicistronic mRNAs is “context-dependent leaky scanning” [13], where the AUG of the first ORF is in a suboptimal configuration, and so a population of ribosomes fail to initiate at this AUG and continue scanning the mRNA to initiate translation at the AUG of the second ORF

Summary

Introduction

Identified [4], and a non-viral dicistronic mRNA, with proposed IRES between the cistrons, has been recognized as an alternative transcript from the Adh locus in Drosophila [5]. A screen of the Drosophila genome data bases for possible dicistronic mRNAs yielded a number of single transcripts encoding two ORFs. Many were like the Adh locus with at least the possibility that IRES sequences might be present. The stoned locus encodes a single transcript of 8.4 kb, and when cDNAs corresponding to this transcript were isolated, it was observed that they included two tandemly arranged ORFs of 2.5 and 3.7 kb (known as stnA and stnB) with a 55-bp intercistronic region (ICR) [14]. Both ORFs have been shown to be translated in vivo [14]. This data is used to interpret the phenotype associated with a mutation at the stoned locus that inserts a methionine in place of a lysine at position 35 in the stnA ORF

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