ABSTRACT: BACKGROUND: Across the fully sequenced microbial genomes there are thousands of examples of overlapping genes. Many of these are only a few nucleotides long and are thought to function by permitting the coordinated regulation of gene expression. However, there should also be selective pressure against long overlaps, as the existence of overlapping reading frames increases the risk of deleterious mutations. Here we examine the longest overlaps and assess whether they are the product of special functional constraints or of erroneous annotation. RESULTS: We analysed the genes that overlap by 60 bps or more among 338 fully-sequenced prokaryotic genomes. The likely functional significance of an overlap was determined by comparing each of the genes to its respective orthologs. If a gene showed a significantly different length from its orthologs it was considered unlikely to be functional and therefore the result of an error either in sequencing or gene prediction. Focusing on 715 co-directional overlaps longer than 60 bps, we classified the erroneous ones into five categories: i) 5'-end extension of the downstream gene due to either a mispredicted start codon or a frameshift at 5'-end of the gene (409 overlaps), ii) fragmentation of a gene caused by a frameshift (163), iii) 3'-end extension of the upstream gene due to either a frameshift at 3'-end of a gene or point mutation at the stop codon (68), iv) Redundant gene predictions (4), v) 5' & 3'-end extension which is a combination of i) and iii) (71). We also studied 75 divergent overlaps that could be classified as misannotations of group i). Nevertheless we found some convergent long overlaps (54) that might be true overlaps, although an important part of convergent overlaps could be classified as group iii) (124). CONCLUSIONS: Among the 968 overlaps larger than 60 bps which we analysed, we did not find a single real one among the co-directional and divergent orientations and concluded that there had been an excessive number of misannotations. Only convergent orientation seems to permit some long overlaps, although convergent overlaps are also hampered by misannotations. We propose a simple rule to flag these erroneous gene length predictions to facilitate automatic annotation.

ABSTRACT: BACKGROUND: Although the overlap of transcriptional units occurs frequently in eukaryotic genomes, its evolutionary and biological significance remains largely unclear. Here we report a comparative analysis of overlaps between genes coding for well-annotated proteins in five metazoan genomes (human, mouse, zebrafish, fruit fly and worm). RESULTS: For all analyzed species the observed number of overlapping genes is always lower than expected assuming functional neutrality, suggesting that gene overlap is negatively selected. The comparison to the random distribution also shows that retained overlaps do not exhibit random features: antiparallel overlaps are significantly enriched, while overlaps lying on the same strand and those involving coding sequences are highly underrepresented. We confirm that overlap is mostly species-specific and provide evidence that it frequently originates through the acquisition of terminal, non-coding exons. Finally, we show that overlapping genes tend to be significantly co-expressed in a breast cancer cDNA library obtained by 454 deep sequencing, and that different overlap types display different patterns of reciprocal expression. CONCLUSIONS: Our data suggest that overlap between protein-coding genes is selected against in Metazoa. However, when retained it may be used as a species-specific mechanism for the reciprocal regulation of neighboring genes. The tendency of overlaps to involve non-coding regions of the genes leads to the speculation that the advantages achieved by an overlapping arrangement may be optimized by evolving regulatory non-coding transcripts.