Pervasive functional translation of noncanonical human open reading frames

Area of Science:

• Molecular Biology
• Genomics
• Human Physiology

Background:

• Ribosome profiling indicates translation occurs beyond canonical coding sequences (CDSs), but these are largely uncharacterized.
• The functional significance and cellular roles of these non-canonical translations remain largely unknown.

Purpose of the Study:

• To systematically identify and characterize non-canonical coding sequences (CDSs) and their encoded microproteins in human cells.
• To investigate the cellular functions, localization, and interactions of these newly identified microproteins.

Main Methods:

• Utilized a systematic CRISPR-based screening strategy to identify non-canonical CDSs.
• Performed functional characterization including cellular localization, protein-protein interaction studies, and analysis of presentation by the human leukocyte antigen (HLA) system.

Main Results:

• Identified hundreds of essential non-canonical CDSs crucial for cellular growth.
• Disruption of these CDSs led to specific transcriptomic and phenotypic changes.
• Characterized microproteins encoded by these CDSs, revealing distinct localizations, binding partners, and HLA presentation.
• Discovered microproteins encoded in upstream open reading frames (uORFs) forming functional bicistronic operons with canonical proteins.

Conclusions:

• Human cells utilize a significant number of functional microproteins encoded outside canonical CDSs.
• These microproteins play critical and diverse roles in cellular functions, including protein complex formation and immune surveillance.
• The identification of functional bicistronic operons in mammals expands our understanding of gene regulation and protein production.