Alternative ORFs and small ORFs: shedding light on the dark proteome

Abstract

Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored 'dark proteome' is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.

Figure 1.

Architecture of alt-ORFs discovered on messenger RNAs (A) and sORFs encoded on transcripts denoted ‘noncoding’ RNAs (B). In (A), the organization of the alt-ORFs (blue) relative to the main ORFs (dark gray) is shown for mature mRNAs with alt-ORFs in the same frame (left), which can result in N-terminal extensions or deletions, and out-of-frame (right) relative to the annotated main-ORF. Annotated start codons for the main-ORF are marked with solid green triangles and alternative start codons are marked with empty green triangles.

Figure 2.

Regulatory roles of alt-ORFs in gene expression. The translation of alt-ORFs has wide-ranging impacts on gene expression. In bacteria, due to coupled transcription-translation, uORF translation may alter downstream RNA structures that impact transcription. A typical example is the trp operon. Uni-directional mRNA translation also can result in a direct effect of uORF translation on downstream main-ORF translation. Possible mechanisms include altering mRNA structures coupled to downstream translation, as well as regulation via leaky scanning and translation re-initiation. uORF translation also can affect mRNA stability throughout the recruitment of components of mRNA surveillance pathways such as UPF1.

Figure 3.

Biological functions of sORF products. The general functions of several small membrane proteins are illustrated. The small proteins can be subunits of a larger protein complexes (such as the CydX, CcoQ and CcoM protein components of cytochrome oxidase complexes in bacteria), affect protein stability (such as the low-magnesium induced MgtS protein that stabilizes the E. coli MgtA magnesium importer) or function (such as the Sarcolipin, Phospholamban, Myoregulin and DWORF modulators of the mammalian CIRCA calcium pump) that mediates membrane reorganization (such as the Minion-Myomerger-Myomixer small protein in skeletal muscle cells) and recruit complexes to cell membranes (such as the Prli42 and SpoVM proteins in L. monocytogenes and B. subtilis, respectively).

Figure 4.

Regulation of alt-ORF translation in eukaryotes. Translation of alt-ORFs is modulated by both cis-sequence elements and trans-acting factors. Stress signaling pathways lead to phosphorylation of eIF2α, which triggers translation re-initiation at a second uORF by affecting the eIF2-GTP-tRNA_i^Met ternary complex (TC) availability. This can block translation of the downstream main-ORF. In response to certain stress conditions, eIF2A promotes non-AUG translation (CUG shown here), which appears to stimulate main ORF translation. mRNA modification in the form of m⁶A may influence alternative start codon selection by slowing ribosome scanning. Additionally, translation of some uORFs is sensitive to intracellular metabolites whose levels are controlled by enzymes encoded by the downstream main-ORF.