In this paper, we present an unusual orientation of protein-coding genes in the S. cerevisiae genome, identifying a previously overlooked gene, NAG1, nested antisense and opposite another protein-coding gene, YGR031W. This gene superstructure represents an evolutionary unit conserved among many fungal species. The strongly conserved YGR031W gene encodes a mitochondrial protein, while NAG1 encodes a 19-kDa membrane protein localized to the yeast cell periphery. To study NAG1 function, we constructed a point mutation disrupting NAG1 but silent with respect to YGR031W; this mutant exhibited hypersensitivity to calcofluor white and altered transcript levels for a significant subset of genes mediating cell wall biogenesis. Furthermore, Nag1p levels were increased upon calcofluor white treatment and reduced in strains deleted for SLT2 and RLM1, key components of the yeast MAPK cell wall integrity pathway. Collectively, this study highlights a role for Nag1p in maintaining yeast cell wall integrity and function, while validating the protein-coding potential of this nested gene.
In particular, the nested organization of genes at the NAG1
locus holds interesting evolutionary implications. Overlapping genes have been found commonly in viruses and microorganisms, where this type of interleaved and nested gene organization presumably contributes to the maintenance of a compact genome—a beneficial characteristic, since genome size in these organisms is limited by the size of the viral particle or cell (22
). Eukaryotic genomes, of course, do not face this constraint, and in this light, two points regarding NAG1
are noteworthy. First, putative NAG1
orthologs are exclusively found opposite an ortholog of YGR031W. Second, YGR031W encodes a mitochondrial protein. Mitochondria are thought to have evolved from purple non-sulfur bacteria (1
), wherein this type of nested gene organization might not be uncommon. Extrapolating from this, we can speculate that the NAG1
locus may represent the remnants of an ancient genetic unit, possibly even tracing back to symbiont gene transfer during mitochondrial evolution. YGR031W is strongly conserved in prokaryotes and eukaryotes alike, but over evolutionary time, NAG1
function may have been lost in organisms lacking a cell wall. Consistent with this possibility, putative NAG1
orthologs are present only in prokaryotes and fungi (Fig. ), although further studies would be necessary to determine whether these orthologs are functional.
The cell wall-related function of Nag1p is supported by three lines of evidence. First, and most striking, is the fact that the nag1-1 mutation leads to a significant decrease in the expression of a large set of cell wall genes during vegetative growth. This is opposite to the more common phenomenon whereby deletion of a cell wall gene causes upregulation of cell wall gene transcription to compensate for the resulting cell wall defects. The negative effect of nag1-1 on cell wall gene expression is more consistent with Nag1p functioning as a type of regulatory protein as opposed to having a direct role in cell wall structure or biosynthesis. This analysis is further supported by the relatively mild cell wall phenotype displayed by the nag1-1 mutant. However, it is important to note that mutation of a number of cell wall-related genes causes calcofluor white hypersensitivity as their only discernible cell wall phenotype; therefore, this second set of observations supporting a cell wall role for Nag1p is consistent with other bona fide cell wall proteins.
Third, the effect of cell wall stress and the cell wall integrity MAPK signaling pathway on NAG1 expression provides compelling support for the cell wall-related function of Nag1p. As is the case for many cell wall-related genes, cell wall stress, such as calcofluor white treatment, induces a modest increase in Nag1p levels. NAG1 may share its promoter region with GSC2 (Fig. ), the stress-inducible subunit of 1,3-β-glucan synthase, and therefore, NAG1 expression could be regulated by processes that also regulate GSC2. Indeed, this region contains a consensus binding site for Rlm1p, a transcription factor regulated by the cell wall integrity MAPK signaling pathway. Since the Rlm1p binding site is palindromic, it should control transcription of appropriately oriented ORFs on either the Watson or Crick strand.
Consistent with this analysis, NAG1
expression is dependent on both Slt2p and Rlm1p in a significant but not exclusive fashion. Intriguingly, the effect of Slt2p and Rlm1p on NAG1
expression is quite apparent during vegetative growth. Although the cell wall integrity pathway is more commonly thought of as a stress response cascade, it is activated during specific periods of the cell cycle (26
). Therefore, NAG1
expression may be controlled through basal signaling of the cell wall integrity pathway. We speculate that this pattern of expression may relate to the positive effect of Nag1p on the transcription of other cell wall genes during vegetative growth. Obviously, a more extensive characterization of Nag1p will be required to confirm this assertion. However, it is clear from our data that Nag1p is a functional protein involved in yeast cell wall integrity.
Here, we have referred to NAG1
as being unique, but, in fact, NAG1
may actually represent the first identified gene of a larger class: the potential certainly exists for other nested antisense genes in yeast. By transposon mutagenesis using a simple gene trap reporter, we previously identified a set of at least 54 putative nested genes in yeast (24
). While we expect that some, and perhaps the majority, of these nested ORFs do not encode protein, additional studies may uncover other nested protein-coding genes previously overlooked in the yeast genome. These overlooked genes potentially represent a wealth of unexplored yeast biology, with implications impacting gene predictions and gene-finding studies in other eukaryotes as well. As a result, the example set by NAG1
may prove useful in refining annotation efforts applied to other genomes, separate from the relevance of this gene as an interesting component of the signaling pathways and networks contributing to yeast cell surface biology.