Tiny but mighty: Diverse functions of uORFs that regulate gene expression

Abstract

Upstream open reading frames (uORFs) are critical cis-acting regulators of downstream gene expression. Specifically, uORFs regulate translation by disrupting translation initiation or mediating mRNA decay. We herein summarize the effects of several uORFs that regulate gene expression in microbes to illustrate the detailed mechanisms mediating uORF functions. Microbes are ideal for uORF studies because of their prompt responses to stimuli. Recent studies revealed uORFs are ubiquitous in higher eukaryotes. Moreover, they influence various physiological processes in mammalian cells by regulating gene expression, mostly at the translational level. Research conducted using rapidly evolving methods for ribosome profiling combined with protein analyses and computational annotations showed that uORFs in mammalian cells control gene expression similar to microbial uORFs, but they also have unique tumorigenesis-related roles because of their protein-encoding capacities. We briefly introduce cutting-edge research findings regarding uORFs in mammalian cells.

Keywords: Gene expression and regulation; Microbes; Stress response; Transcription; Translation; UORFs.

Graphical abstract

Fig. 1

Illustration of different uORF types. (1) Non-overlapping uORFs, (2) multiple uORFs, (3) out-of-frame overlapping uORFs, and (4) in-frame overlapping uORFs.

Fig. 2

Translation initiation in eukaryotes. A complete translation process includes translation initiation, elongation, termination, and ribosome recycling. During translation initiation, the 40S subunit binds to the ternary complex (TC) consisting of eIF2, GTP, and methionine initiator transfer RNA (Met-tRNA_i^Met) along with eIF5 to form the 43S PIC. The binding of PIC to mRNA is facilitated by the eIF4 complex. The eIF4 complex, which consists of the cap-binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A, recruits ribosomes to mRNA. The mRNA-binding PIC then slides downstream of the mRNA, scanning for a start codon. After the translation start site is selected, eIF2 promotes GTP hydrolysis and GDP dissociates from mRNA. The 60S subunit is then recruited to PIC to form the 80S initiation complex (IC) for elongation. During elongation, the ribosome moves along the mRNA to synthesize the nascent peptide according to each codon. Translation termination occurs when the ribosome encounters a stop codon, after which the newly synthesized polypeptide is released and the ribosome dissociates and is recycled.

Fig. 3

Regulatory functions of uORFs. (A) Leaky scanning. When the uORF start codon is not selected as a translation start site, PIC is expelled from the uORF stop codon and reassembled at the mORF start codon to initiate translation. (B) Ribosome stalling/drop-off. The uORF stop codon mediates uORF translation termination, which leads to the separation of the 40S and 60S subunits from the mRNA and repressed mORF translation. (C) Translation reinitiation. The 40S subunit attaches to the mRNA following uORF translation termination. The 60S subunit is then recruited to the mORF start codon to restart mORF translation. (D) Nonsense-mediated mRNA decay. uORF translation termination at the stop codon triggers nonsense-mediated mRNA decay facilitated by UPF proteins, which is followed by the recruitment of an exonuclease or endonuclease that degrades mRNA. (E) Factors contributing to uORF inhibitory efficiency. The start codon, stop codon, codon usage, uORF length, number of uORFs, Kozak sequence context around the start codon, uORF-encoded peptides, distance from the mORF, and uORF secondary structures contribute to uORF effects on mORFs.

Fig. 4

uORF functions in fungal stress responses. (A) uORFs regulate GCN4 expression. Under normal conditions, uORF1 translation leads to uORF4 translation, which then decreases GCN4 translation because of relatively poor REI. Under amino acid starvation conditions, TC insufficiency hampers 40S ribosomal subunit recruitment and translation reinitiation at uORF4, causing the ribosome to bypass uORF4 and reinitiate translation at the GCN4 start codon. (B) uORFs regulate fil1 expression. Under normal conditions, fil1 expression is repressed by the translation of uORF4 and 5. In response to amino acid starvation, decreased TC levels cause the 40S ribosomal subunit to bypass repressive uORF4 and 5, thereby mediating fil1 translation. (C) uORFs regulate Cpa1 expression. When the arginine concentration is low, leaky scanning suppresses uORF translation and promotes Cpa1 expression. Conversely, high arginine levels induce nascent peptide conformational changes that lead to ribosome stalling and inhibit Cpa1 translation.

Fig. 5

Regulatory effects of uORFs in bacteria. In the absence of antibiotics, the whiB7/wblC uORF is completely translated, which suppresses whiB7/wblC transcription and promotes Rho-independent terminator (RIT) formation, resulting in premature transcriptional termination. Antibiotic stress conditions inhibit uORF translation by favoring anti-terminator formation, which is conducive to whiB7/wblC transcription.

Fig. 6

Regulatory effects of uORFs in viruses. (A) Ribosome shunting in CaMV. After the first uORF is translated, the ribosome moves directly to the region downstream of the looped structure on the 35S leader, bypassing the regions in the stem structure. (B) uORF-mediated translation regulation in MHV. Under normal conditions, the uORF in the SL4b stem-loop is translated and ORF1 translation is attenuated. A mutation in the uORF within SL4b inhibits uORF translation, but increases ORF1 translation.