Impacts of uORF codon identity and position on translation regulation.

Yizhu Lin,Hunter Kready,Gemma E May,Lauren Nazzaro,Mao Mao,C Joel Mcmanus,Yehuda Creeger,Pieter Spealman

doi:10.1093/nar/gkz681

Abstract

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5′ mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

Highlights

As an essential step in gene expression, mRNA translation is highly regulated by complex interactions between cis-acting sequences and trans-acting factors
We tested the effects of thousands of YAP1 Upstream open reading frames (uORFs) sequence variants on gene expression using fluorescence-associated cell n sorting (FACS)-uORF (Figure 1A), a high-throughput uORF functional assay inspired by massively parallel reporter assays of gene expression [15,24,25]
Coding sequence variation creates a wide range of uORF effects on main ORF expression Having established FACS-uORF, we investigated the impact of codon variants on uORF function

Summary

Introduction

As an essential step in gene expression, mRNA translation is highly regulated by complex interactions between cis-acting sequences and trans-acting factors. Pre-initiation complexes (PICs), comprised of 40S small ribosomal subunits and numerous initiation factors, assemble at mRNA 5 ends and scan the mRNA in a 5 to 3 direction until a suitable start codon is encountered [1]. As a consequence of directional scanning, the presence of mRNA structures and regulatory sequences between the 5 7-meG cap and the main ORF coding sequence can affect initiation efficiency. Upstream open reading frames (uORFs) are short coding sequences that function as potent cis-acting regulators of translation initiation. While most studied uORFs repress translation at downstream main ORF (mORF) coding sequences, some act as stress-dependent enhancers through re-initiation at downstream mORF start codons [2,3]

Methods

Results

Conclusion