BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 fying selection. These sequences are thus of 'intermediate' character, i.e., they have evidence of both protein-coding ability and pseudogenicity. Antisense homologies of human pseudogenes to other full- length human cDNAs Transcribed pseudogenes can regulate the expression of other genes by RNA interference mechanisms. For exam- ple, antisense transcribed RNA from a NOS pseudogene regulates the expression of neural nitric oxide synthase (nNOS) protein through formation of RNA duplex [34,35]. Therefore, we investigated how many of the TPAs have antisense homology to the annotated full-length human cDNAs (E-value < 1e-10 and alignment length > = 100 nucleotides). A small proportion (8.3%, 69/828) of the human (VEGA) pseudogenic transcripts have either complete or partial, but significant, reverse complement (antisense) homology to human cDNAs. Of these, 63 have short length strong antisense homology to human cDNAs (alignment length > = 20, mismatches < = 2). However, there is no significant association of such anti- sense homologies to pseudogene transcription, since non- transcribed pseudogenes have similar levels of antisense homology (7.65%, χ2 test P-value = 0.5). Out of the identified 68 human conserved TPAs, 3 have antisense homology to human cDNAs (E-value < 1e-10, 5 if alignment length > = 50 nucleotides is considered) (Table 3). These are cases that may generate small interfer- ing RNAs (siRNAs) that could potentially regulate the expression levels of their homologous genes. Pseudogenes have been implicated in the negative regulation of paren- tal genes (for a review, see ref. [2]) and in the Dicer-medi- ated generation of small RNAs [18,19]. It would be interesting to verify experimentally whether these pseudo- gene transcripts can indeed generate small interfering RNAs, through the action of Dicer. Small RNA mappings to pseudogenes Transcribed pseudogenes can also regulate the transcrip- tion of genes by producing siRNAs that have antisense homology [18,19]. Due to unavailability of genome-wide human siRNA data, we used the siRNA data for the mouse genome from Tam et al. [19] and Watanabe et al. [18] to check how many of the small RNAs mapped to mouse transcribed pseudogenes that we identified. Interestingly, 24 out of 136 (17.6%) mouse TPAs had siRNA mappings compared to ~1% (178/18168) of the total mouse pseu- dogenes. The above difference is statistically significant (P-value < 0.05, using normal statistics for the distribu- tion of the mean number of transcribed pseudogenes in a sample of 136 cases). This demonstrates that transcribed pseudogenes are significantly likely to generate siRNAs in mouse. For comparison, in Arabidopsis thaliana, ~40% of 572 pseudogenes have small RNA mappings [36]. Conclusion In this study, we identified hundreds of cases of putative transcribed pseudogene annotations (TPAs), in the human genome. Importantly, we detected evidence for selection pressure on these transcribed elements. These findings therefore draw wider attention towards the potential functionality of these genomic elements. In addition, we found that 68 human TPAs are conserved in at least 2 other studied mammals. These human TPAs have ancient origins dating back >120 million years ago, as evidenced by their conservation patterns across dis- tantly related mammals. These pseudogenes represent novel genomic elements of potential functional relevance. We have shown that human TPAs that are syntenically conserved in rhesus monkey generally behave like non- protein-coding sequences, despite significant selection pressure on them, relative to the surrounding genomic DNA. Examination of Ka/Ks values for TPAs that are con- served in more divergent species (mouse and dog), indi- cated that some TPAs might actually be protein-coding. However, we cannot rule out other reasons for these low Ka/Ks values. For example, it is possible that some of these sequences had phases of protein-coding ability at some evolutionary stage. Also, it is possible that there is an imprint of purifying selection on these sequences because of selection pressure to form small interfering RNAs with homologous protein-coding genes. Ultimately, these Table 3: Human conserved TPAs that have antisense homology to human full length cDNAs. Pseudogene/transcript id OTTHUMT00000269970 OTTHUMT00000270027 chr10_Q96RG0.4_- urn:lsid:pseudogene.org:9606.Pseudogene:18315 ENST00000344386 OTTHUMT00000082689 ENST00000343936 94.92 87.59 83.23 96.3 91.38 ENSEMBL transcript id Antisense identity (%) Alignment length E-value ENST00000323294 ENST00000379565 ENST00000315032 118 282 161 81 58 7.00e-46 2.00e-70 1.00e-17 3.00e-31 2.00e-12 Page 10 of 14 (page number not for citation purposes)