Positive natural selection of N6-methyladenosine on the RNAs of processed pseudogenes

Canonical nonsense-mediated decay (NMD) is an important splicing-dependent process for mRNA surveillance in mammals. However, processed pseudogenes are not able to trigger NMD due to their lack of introns. It is largely unknown whether they have evolved other surveillance mechanisms.

Here, we find that the RNAs of pseudogenes, especially processed pseudogenes, have dramatically higher m⁶A levels than their cognate protein-coding genes, associated with de novo m⁶A peaks and motifs in human cells. Furthermore, pseudogenes have rapidly accumulated m⁶A motifs during evolution. The m⁶A sites of pseudogenes are evolutionarily younger than neutral sites and their m⁶A levels are increasing, supporting the idea that m⁶A on the RNAs of pseudogenes is under positive selection. We then find that the m⁶A RNA modification of processed, rather than unprocessed, pseudogenes promotes cytosolic RNA degradation and attenuates interference with the RNAs of their cognate protein-coding genes. We experimentally validate the m⁶A RNA modification of two processed pseudogenes, DSTNP2 and NAP1L4P1, which promotes the RNA degradation of both pseudogenes and their cognate protein-coding genes DSTN and NAP1L4. In addition, the m⁶A of DSTNP2 regulation of DSTN is partially dependent on the miRNA miR-362-5p.

Our discovery reveals a novel evolutionary role of m⁶A RNA modification in cleaning up the unnecessary processed pseudogene transcripts to attenuate their interference with the regulatory network of protein-coding genes.