Rp3: Ribosome profiling-assisted proteogenomics improves coverage and confidence during microprotein discovery

Area of Science:

• Genomics
• Proteomics
• Molecular Biology

Background:

• The protein-coding genome is expanding with the discovery of non-canonical translation.
• Ribosome profiling (Ribo-Seq) is key for identifying small Open Reading Frames (smORFs) encoding microproteins.
• Current Ribo-Seq methods struggle with multi-mapping reads and identifying protein evidence for smORFs.

Purpose of the Study:

• To develop a novel computational pipeline for improved microprotein identification.
• To overcome limitations of standard Ribo-Seq in detecting smORFs.
• To integrate proteogenomics with Ribo-Seq for robust microprotein evidence.

Main Methods:

• Developed the Rp3 pipeline, integrating proteogenomics and Ribo-Seq data.
• Applied Rp3 to analyze ribosome-protected footprints (RPFs).
• Focused on unambiguous RPF mapping to identify smORFs.

Main Results:

• Rp3 successfully identified microproteins missed by conventional Ribo-Seq pipelines.
• The pipeline maximizes proteomics detection and confidence for microprotein-encoding smORFs.
• Rp3 provides unambiguous evidence for novel coding sequences.

Conclusions:

• Rp3 enhances the discovery and validation of microproteins.
• The integration of proteogenomics and Ribo-Seq is crucial for expanding the annotated genome.
• This approach addresses key challenges in smORF identification and protein evidence.