Characterizing the cis-regulatory roles of translating downstream open reading frames

Abstract

Understanding the coding potential of the genome is a fundamental step for deciphering the molecular framework of living systems. Traditionally, eukaryotic mRNAs were thought to contain only one protein-coding sequence. However, with the advent of high-throughput techniques like ribosome profiling and proteomics, people have identified small translated open reading frames (ORFs) in both the 5′ UTRs and 3′ UTRs called upstream ORFs (uORFs) and downstream ORFs (dORF), respectively. The lab is mainly interested in newly found translation events in 3′ UTR and the unexplored consequences of the presence of actively translating ribosomes in this region. The study explores how the translation of dORFs influences other regulatory processes involving 3′ UTRs. Using an in vitro transcribed mCherry-based reporter system, we investigated the impact of ribosomes translating poly-A sequences on mRNA stability and protein output. We hypothesized that the translation of the poly-A sequence imposed by dORF would trigger transcript decay by the No-Go Decay (NGD) mechanism. To test this, we measured the protein levels as well as transcript levels. Our findings suggest that the translation of multiple adenines within dORFs does not induce large-scale mRNA decay. Furthermore, we also explored the interaction between the active translating ribosomes and repression by microRNA-AGO complex. We hypothesize that dORFs work as negative regulators of microRNA (miRNA) targeting, such as observed in canonical ORFs, where miRNA target sites are not efficiently responsive to their cognate miRNA. To test this, we designed a mCherry expression vector containing an artificial 3′ UTR with a dORF encompassing two miRNA target sites for miR-1. Protein levels of mCherry were determined by cytometry analysis, and levels of transcripts were determined by qPCR.