Identification of microprotein-coding intronic polyadenylation isoforms and function in genotoxic anticancer drug response

Background: Many transcript isoforms generated by intronic polyadenylation (IPA) encode isoforms of canonical proteins. Microproteins are an emerging class of small proteins translated from small open reading frames (sORFs) in noncoding RNAs and mRNAs, but their production by IPA isoforms is unknown. Results: Here, by crossing 3′-seq, Ribo-Seq, and mass-spectrometry data, we identify 297 genes with a microprotein-coding IPA isoform terminating in a 5′UTR intron (coined miP-5′UTR-IPA isoform). By 3′-seq and long-read RNA-seq analyses in lung cancer cells treated with cisplatin, a DNA-cross-linking anticancer drug, we find that cisplatin globally favors the expression of (miP-5′UTR-)IPA isoforms relative to full-length mRNAs, mainly by decreasing the latter through an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. The cisplatin-regulated miP-5′UTR-IPA isoform in the PRKAR1B gene is translated, as it is associated with light polysome fractions and contains Ribo-Seq-supported sORFs in its alternative last exon, and the microprotein (PRKAR1B-IPA-miP2) encoded by its sORF#2 is detected by Western blot and immunofluorescence. CRISPR editing of either the IPA site or the sORF#2 initiation site leads to decreased cell growth inhibition by cisplatin and camptothecin, another genotoxic drug. Mechanistically, PRKAR1B-IPA-miP2 promotes p53 protein induction by cisplatin. Finally, 70 miP-5′UTR-IPA isoforms are detected in normal cells, and 143 are upregulated by cisplatin. Conclusions: Here, we show that IPA isoforms are a novel source of microproteins, and we reveal the novel paradigm of miP-5′UTR-IPA genes that produce both a canonical full-length mRNA and a microprotein-coding IPA isoform.