A question of size: the eukaryotic proteome and the problems in defining it

Abstract

We discuss the problems in defining the extent of the proteomes for completely sequenced eukaryotic organisms (i.e. the total number of protein-coding sequences), focusing on yeast, worm, fly and human. (i) Six years after completion of its genome sequence, the true size of the yeast proteome is still not defined. New small genes are still being discovered, and a large number of existing annotations are being called into question, with these questionable ORFs (qORFs) comprising up to one-fifth of the 'current' proteome. We discuss these in the context of an ideal genome-annotation strategy that considers the proteome as a rigorously defined subset of all possible coding sequences ('the orfome'). (ii) Despite the greater apparent complexity of the fly (more cells, more complex physiology, longer lifespan), the nematode worm appears to have more genes. To explain this, we compare the annotated proteomes of worm and fly, relating to both genome-annotation and genome evolution issues. (iii) The unexpectedly small size of the gene complement estimated for the complete human genome provoked much public debate about the nature of biological complexity. However, in the first instance, for the human genome, the relationship between gene number and proteome size is far from simple. We survey the current estimates for the numbers of human genes and, from this, we estimate a range for the size of the human proteome. The determination of this is substantially hampered by the unknown extent of the cohort of pseudogenes ('dead' genes), in combination with the prevalence of alternative splicing. (Further information relating to yeast is available at http://genecensus.org/yeast/orfome)

Figure 1

Number of yeast ORFs as a function of the minimum allowed ORF length. The total number of annotated ORFs in the yeast proteome is plotted against minimum ORF length (continuous blue line). A curve for known proteins or proteins confirmed by homology to a known protein is shown (red line), along with a green curve for the remaining ORFs that have no homology to a known protein (or are not otherwise characterized). Also displayed (dotted pink line) is the total number of additional ‘acceptable’ ORFs from the yeast genome that have good codon adaptation (CAI ≥0.11) that do not overlap an annotated gene or other genomic feature. The plots are cumulative backwardly at intervals of 10 residues.

Figure 2

The variation in the size of the WormPep database over time. The size of the WormPep database is plotted against time for the period after and just prior to publication of the genome sequence. The dotted line indicates the approximate time of genome sequence completion.

Figure 3

Human gene numbers and proteome size. The figure depicts, in bar chart form, the number of human genes (blue bar) from various estimates and a corresponding estimate for proteome size (orange bar). Gene numbers and proteome sizes are shown for the other sequenced eukaryotes, with the same coloring. The size of the human proteome (N_CDS) can be estimated as follows: N_CDS = f₁.f₂.N_genes, where f₁ is the proportion of gene structures that are not pseudogenic, and f₂ is the ratio of the total number of distinct protein-coding transcripts to the total number of genes (arising from alternative splicing). Assuming 0.76 ≤ f₁ ≤ 0.91 (see text), a minimum value of f₂ = 1.16 can be derived from the alternative splicing survey of Mironov et al. (72), and a maximum value f₂ = 2.22 is calculable from the alternative splicing analysis in Lander et al. (56). Using these, and the wider range for N_genes given by Venter et al. (57) a range of approximately 20 300 to approximately 83 800 is yielded for N_CDS. This range is clearly rather large, and is reminiscent of the range of values arising for estimates of N_genes that arose in the months and years prior to publications of the human genome.