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

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Intronic polyadenylation (IPA) generates transcript isoforms encoding canonical proteins.
• Microproteins, translated from small open reading frames (sORFs), are an emerging class of proteins.
• The role of IPA isoforms in microprotein production was previously unknown.

Purpose of the Study:

• To investigate if intronic polyadenylation (IPA) isoforms can produce microproteins.
• To identify and characterize microprotein-coding IPA isoforms.
• To explore the regulation and function of these isoforms, particularly in response to anticancer drugs.

Main Methods:

• Integrated analysis of 3 seq, Ribo-Seq, and mass spectrometry data.
• Long-read RNA-seq and 3 seq in cisplatin-treated lung cancer cells.
• CRISPR editing, Western blot, and immunofluorescence for protein validation.
• Polysome profiling to assess translation.

Main Results:

• Identified 297 genes with microprotein-coding IPA isoforms (miP-5 UTR-IPA isoforms).
• Cisplatin treatment globally favors miP-5 UTR-IPA isoforms over full-length mRNAs by inhibiting transcription processivity.
• Demonstrated translation of a specific miP-5 UTR-IPA isoform (PRKAR1B-IPA-miP2) and its role in promoting p53 induction.
• CRISPR editing of IPA sites or sORF#2 reduced sensitivity to genotoxic drugs.

Conclusions:

• Intronic polyadenylation (IPA) isoforms represent a novel source of microproteins.
• Introduced the concept of miP-5 UTR-IPA genes producing both canonical and microprotein-coding isoforms.
• miP-5 UTR-IPA isoforms and their encoded microproteins play a role in cellular response to genotoxic stress.