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

Abstract

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.

Keywords: Bifunctional genes; Innate immunity; Short open reading frames (sORFs).

Figure 1. Key figure. Discovering and characterizing novel peptides

(A) Steps in discovering translated open read frames (ORFs). Top: determining the transcriptome of the cell type of interest. Middle: Ribo-Seq specifies ribosome-associated ORFs. Additional confidence gained from three-nucleotide periodicity. Bottom: computational analyses including conservation score, identification of Kozak sequences, and predicted protein domains increase confidence in ORF translation. (B) Approaches to high-throughput validation of functional translation. Proteomics: proteins are measured from whole-cell lysate or MHC–peptide complexes. CRISPR-Cas9 Screen: GuideRNAs (gRNAs) against putative short ORFs (sORFs) are sequenced in bulk, and changes in distribution of guides imply sORF-encoded peptide (SEP) function (i.e., disappearance of guides over time, indicating a SEP critical for viability). Perturb-Seq: single-cell RNA sequencing links sORF disruption to phenotype. Homology directed repair (HDR): epitope tag insertion inframe with the sORF via nucleofection with Cas9-complexed RNAs (Box 2). (C) Approaches to validating sORF coding potential and determining SEP function. Coding versus noncoding function: ablating the start codon of the sORF leaves RNA function intact. Alternatively, re-engineering sORF with synonymous mutations alters RNA sequence and structure but leaves SEP intact. Conclusions about the relative importance of SEP versus RNA contribution to phenotype may be context-specific. SEP-protein interactions: a tagged SEP, or an anti-SEP antibody, can be used to pull down the SEP and interaction partners which can be identified by mass spectrometry (MS). Localization: determined by epitope tagging and immunostaining. Split fluorescent systems may reduce disruption of wild-type (WT) SEP activity while providing direct fluorescent confirmation of translation. Abbreviations: LC, liquid chromatography; GFP, green fluorescent protein. Figure generated using Biorender.com.

Figure 2.. Newly characterized short open reading frame (sORF)-encoded peptide (SEP) in innate immunity.

(A) In mouse bone-marrow-derived macrophages (BMDMs), Aw112010 SEP is essential to robust mucosal immunity. In mouse CD4⁺ T cells Aw112010 RNA guides KDM5A demethylase to histones at the II10 locus, reducing expression [8,82]. (B) In K562s and an acute myeloid leukemia (AML) cell line (human), a HOXB-AS3 transcript produces a SEP that antagonistically binds at the hnRNPA1 RNA-binding domain, reducing the amounts of cancer-associated transcripts and decreasing proliferation. In a colon cancer line, an alternative HOXB-AS3 transcript guides DNA methylase EBP1 to the ribosomal DNA locus, increasing transcription and contributing to a proliferative phenotype [10,30]. (C) In BMDMs, 1810058I24Rik produces Mm47, which localizes to the mitochondria and contributes to Nlrp3 inflammasome generation in response to lipopolysaccharide (LPS). LPS also triggers degradation of Mm47, perhaps as a timer on the resolution of inflammation [76]. (D) in BMDMs, MIR155HG produces miPEP155 which antagonistically binds HSC70 and reduces MHCII display [16]. This transcript also serves as a precursor to miRNA miR-155 which regulates many immune-related RNAs. (E) In human monocyte-derived macrophages, NMES produces SEP C15ORF48 which competes with NDUFA4 in binding cytochrome c oxidase (CcO). Additionally, miRNA miR-147b downregulates the NDUFA4 transcript [92]. Figure generated using Biorender.com.