Ribosomal profiling adds new coding sequences to the proteome.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) provides deep insights into genomic elements and their functions.
• Ribosome footprint sequencing (Ribo-Seq) offers single-nucleotide resolution of active translation by sequencing ribosome-bound fragments (RBFs).
• Previous bioinformatic studies identified numerous small open reading frames (smORFs) but lacked experimental validation for their translation.

Purpose of the Study:

• To review methodologies for defining translation from Ribo-Seq data.
• To address challenges in validating translation of novel smORFs within long non-coding RNAs (lncRNAs) and untranslated regions (UTRs).
• To explore bioinformatic and biochemical techniques for corroborating smORF translation.

Main Methods:

• Deep sequencing of ribosome-bound fragments (RBFs) using Ribo-Seq.
• Bioinformatic analysis of putative small open reading frames (smORFs).
• Review of experimental approaches for validating translation.

Main Results:

• Ribo-Seq data indicate that hundreds of putative smORFs in lncRNAs and UTRs are associated with ribosomes, suggesting active translation.
• These findings challenge the established understanding of the genome's protein-coding capacity.
• Novel smORFs are being identified in previously non-coding or regulatory regions.

Conclusions:

• Ribo-Seq is a powerful tool for discovering translated smORFs in eukaryotic genomes.
• Further bioinformatic and biochemical validation is crucial for confirming the functional significance of these novel translated smORFs.
• The discovery of translated smORFs expands our understanding of the transcriptome and proteome.