Trends in Immunology

Trends in Immunology

Volume 43, Issue 9, September 2022, Pages 741-756
Journal home page for Trends in Immunology

Review
Short open reading frame genes in innate immunity: from discovery to characterization

https://doi.org/10.1016/j.it.2022.07.005Get rights and content

Highlights

  • New approaches have implicated hundreds of long noncoding RNAs as potential protein coding genes through overlooked short open reading frames (sORFs).

  • There are many thousands of sORFs, with multiple lines of evidence supporting production of sORF-encoded peptides (SEPs), compiled in databases but uninvestigated.

  • Confirmation of production and functional characterization can be nuanced, but thoughtful interrogation has already expanded the known proteome and our understanding of important biological pathways. This includes contributions to innate immune function in mouse and human.

  • Although the expanding proteome is likely to interest investigators from all fields, there is reason to believe that immunologists are particularly well positioned to make impactful discoveries.

Next-generation sequencing (NGS) technologies have greatly expanded the size of the known transcriptome. Many newly discovered transcripts are classified as long noncoding RNAs (lncRNAs) which are assumed to affect phenotype through sequence and structure and not via translated protein products despite the vast majority of them harboring short open reading frames (sORFs). Recent advances have demonstrated that the noncoding designation is incorrect in many cases and that sORF-encoded peptides (SEPs) translated from these transcripts are important contributors to diverse biological processes. Interest in SEPs is at an early stage and there is evidence for the existence of thousands of SEPs that are yet unstudied. We hope to pique interest in investigating this unexplored proteome by providing a discussion of SEP characterization generally and describing specific discoveries in innate immunity.

Section snippets

Gene annotation and sORFs

Beginning with the sequencing of the yeast Saccharomyces cerevisiae genome in the 1990s, the scientific community has shown considerable interest in comprehensive identification and annotation of protein-coding genes in eukaryotes. Early efforts focused on ATG-initiated ORFs capable of encoding a polypeptide of at least 100 amino acids [1,2]. ORFs that did not meet this length cut-off, short open reading frames (sORFs) (see Glossary), required additional evidence to merit a protein-coding

Sequence analysis

Discovering new SEPs begins with sequence analysis. The standard scanning model of translation initiation involves a preinitiation complex binding the RNA 5′-cap and traversing the transcript until reaching a Kozak sequence centered at the start codon AUG. There, the remaining translational machinery engages, and elongation of the polypeptide occurs. Once a stop codon is encountered, translation is terminated, and the ribosome dissociates from the transcript [15]. This model implies a simple

Candidate validation, approaches, and drawbacks

Translated sORF predictions based on ribosomal association likely misestimate the coding potential of transcripts and say nothing about the function of predicted SEPs (including whether they are functional at all). To confirm novel peptide production, candidate sORFs are typically validated via peptide tagging and microscopy or immunoprecipitation (IP). SEPs frequently influence phenotype by complexing with larger protein partners [16,17,70., 71., 72.]; therefore, determining these partners

High-throughput validation

Individual, high-resolution characterization of SEPs will be an important part of correctly annotating the genome and characterizing SEP–protein interaction networks. However, the large numbers of putatively coding sORFs detected from Ribo-Seq and sequence analysis argues for the application of high-throughput methods to validate translation en masse. Broadly, there are two approaches: peptidomics via MS (Box 1) and genome editing with CRISPR-Cas (Box 2). In both cases the challenge is for the

An emerging class: bifunctional genes

Here we describe SEPs that were recently discovered and characterized in innate immune (and innate immune-derived) cell lines. We also note instances where the RNA itself is known to contribute to a phenotype distinctly from the SEP; this is the case in four of the five examples shown in Figure 2. Although the sample size is too small to make strong inferences, the high representation of these ‘coding-noncoding’ or ‘bifunctional’ [85,86] genes suggests that future studies of SEPs would do well

Concluding remarks

Nuanced biomolecular interrogation has allowed researchers to differentiate between SEP and RNA activity, and there are many databases containing thousands of sORFs and lncRNAs that are yet to be investigated (Table 2). Immunologists might find the study of this expanded proteome particularly fruitful. It is well established that the transcriptome is drastically changed under conditions of inflammatory stimuli. Furthermore, it is reported that multiple components of translation initiation

Acknowledgments

S.C. is supported by R01AI148413 from National Institute of Allergy and Infectious Diseases and R35GM137801 from the National Institute of General Medical Sciences. E.M. is supported by T32HG012344 and in part by R35GM137801.

Declaration of interests

S.C. is a paid consultant to NextRNA Therapeutics. No interests are declared by E.M.

Glossary

Dark proteome
understudied and under-characterized proteins and peptides, including those that arise from UTRs and noncoding RNAs.
FASTQs
the standard short-read sequencing format for bioinformatic sequencing analysis.
Homology-directed repair (HDR)
repair of DNA breaks using a homologous template, allowing the insertion of genetic material.
Lipopolysaccharide (LPS)
a PAMP component of Gram-negative bacterial cell walls. In its purified form, it is commonly used as an inflammation-inducing ligand in

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