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

Author:

Vieira de Souza EduardoORCID,L. Bookout Angie,Barnes Christopher A.ORCID,Miller Brendan,Machado Pablo,Basso Luiz A.,Bizarro Cristiano V.ORCID,Saghatelian AlanORCID

Abstract

AbstractThere has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs.

Publisher

Springer Science and Business Media LLC

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