Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome

AbstractPseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. When transcribed, pseudogenes may encode proteins or enact RNA-intrinsic regulatory mechanisms. However, the extent, characteristics and functional relevance of the human pseudogene transcriptome are unclear. Short-read sequencing platforms have limited power to resolve and accurately quantify pseudogene transcripts owing to the high sequence similarity of pseudogenes and their parent genes. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes. Pseudogene transcripts are expressed in tissue-specific patterns, exhibit complex splicing patterns and contribute to the coding sequences of known genes. We survey pseudogene transcripts encoding intact open reading frames (ORFs), representing potential unannotated protein-coding genes, and demonstrate their efficient translation in cultured cells. To assess the impact of noncoding pseudogenes on the cellular transcriptome, we delete the nucleus-enriched pseudogene PDCL3P4 transcript from HAP1 cells and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the transcriptional landscape underpinning human biology and disease.