Regulation of the Expression of Ribosomal Proteins by Small Downstream ORFs

Abstract

Eukaryotic mRNAs have historically been believed to harbour a single open reading frame (ORF). However, over the last decade, ribosome profiling has revealed extensive ribosome occupancy in the untranslated regions (5′- and 3′UTRs), overturning the “one mRNA-one protein” dogma. Similar evidence has been reported in mass spectrometry-based studies, too, indicating diversification of the known proteome. Translation in the 3′UTR of mRNAs can occur due to stop codon readthrough or independent translation initiation at a downstream ORF (dORF). While stop codon readthrough results in a C-terminally extended protein isoform, independent translation initiation generates unique polypeptide(s) encoded by the 3′UTR. Both these phenomena are observed across all domains of life. Although translation of dORFs is known to regulate the expression of the canonical main ORF, the mechanism of this regulation is still elusive. We looked for evidence of translation events in the 3′UTRs of ribosomal protein transcripts. Analyses of ribosome profiling data and the evolutionary conservation of these transcripts suggested that 3′UTRs of human RPL36A (L36A or eL42) and RPS27A (S27A or eS31) mRNAs may undergo translation. Further experimental analyses using luminescence-based assays, qRT-PCRs, fluorescence microscopy, and western blotting validated that both L36A and S27A encode a translatable short ORF in their 3′UTRs, which generates a microprotein. The presence of the dORF increases transcript levels, leading to increased L36a and S27a protein expression, as observed using qRT-PCR and western blotting. To investigate the mechanism of regulation, we looked for alternative polyadenylation sites, binding sites of known RNA-binding proteins, m6A sites, and miRNA-binding sites in and around the dORF region of L36A and S27A using a combination of computational and experimental approaches. Following a computational analysis, we established that a miRNA-based regulation could be present for L36A mRNA. Mutating miRNA-binding sites in the L36A mRNA resulted in the upregulation of L36A mRNA levels even in the dORF-mutated background. We checked the expression levels of the miRNAs to focus on the miRNA of interest. Performing qRT-PCR after treating cells with specific miRNA mimics and inhibitors helped us delineate that hsa-miR-5701, which binds to the L36A mRNA, overlapping with the dORF, regulates L36A mRNA levels. This suggests that in the presence of the dORF, due to ribosome occupancy in the dORF for its translation, the miRNA machinery fails to bind the mRNA, resulting in the L36A canonical ORF being translated at normal levels. However, when the dORF is mutated, miRNAs can bind and cause translation repression and/or mRNA degradation of the L36A mRNA, as ribosomes do not occupy the dORF to hinder miRNA binding, leading to decreased L36a expression. Similarly, CRISPR-based deletion of the dORF of L36A and S27A resulted in decreased mRNA levels of the transcript. The L36A deletion clones also demonstrated slow proliferation and reduced global translation compared to parental wild-type cells. This study highlights a unique mechanism of ribosome formation and translation in mammalian cells by demonstrating a similar mechanism of expression regulation of two ribosomal protein transcripts.