Discovering microproteins: making the most of ribosome profiling data

Abstract

Building a reference set of protein-coding open reading frames (ORFs) has revolutionized biological process discovery and understanding. Traditionally, gene models have been confirmed using cDNA sequencing and encoded translated regions inferred using sequence-based detection of start and stop combinations longer than 100 amino-acids to prevent false positives. This has led to small ORFs (smORFs) and their encoded proteins left un-annotated. Ribo-seq allows deciphering translated regions from untranslated irrespective of the length. In this review, we describe the power of Ribo-seq data in detection of smORFs while discussing the major challenge posed by data-quality, -depth and -sparseness in identifying the start and end of smORF translation. In particular, we outline smORF cataloguing efforts in humans and the large differences that have arisen due to variation in data, methods and assumptions. Although current versions of smORF reference sets can already be used as a powerful tool for hypothesis generation, we recommend that future editions should consider these data limitations and adopt unified processing for the community to establish a canonical catalogue of translated smORFs.

Research on protein and peptides in scorpion based on transcriptomics- proteomics-peptidomics integrated association analysis strategy

“…Subsequently, we established the gene database of scorpion through transcript assembly and ORF prediction [23][24] , and obtained a total of 17605 gene sequences and 12368 proteins in the sample. The process of eukaryotic Transcription initiation is very complex and often requires the assistance of multiple protein factors.…”

Small but mighty: the rise of microprotein biology in neuroscience

“…There is currently a lack of standardization in the experimental methods, data quality control, and analysis of sORFs and microproteins, which has led to significant variability in the identification of translated sORFs. Given the need to adopt rigorous, uniform standards for microprotein validation, several groups have proposed consensus definitions to improve the reliability and consistency of ncORF and protein coding identification (Mudge et al, 2022 ; Chothani et al, 2023 ; Prensner et al, 2023 ). These definitions include the independent identification of a sORF across multiple studies, detection by multiple experimental methods (e.g., Ribo-seq plus mass spectrometry, epitope tagging and western blot, or detection by endogenous antibodies), and/or the presence within the microprotein of disease-associated mutations ( Table 1 ).…”