Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells.

Joana Silva,Luísa Romão,Pedro Nina

doi:10.3390/biomedicines9080911

Joana Silva, Luísa Romão + Show 1 more

Open Access

https://doi.org/10.3390/biomedicines9080911

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Abstract

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

Highlights

Gene expression is extremely regulated at the level of mRNA translation, especially at its initiation step, where the initiation codon is identified and decoded [1,2]
Vanderperre and co-workers predicted bioinformatically the presence of two AUG upstream open reading frames (uORFs) within the ATP-binding cassette subfamily E member 1 (ABCE1) 50 -leader sequence [63], those corresponding to uORF2 and uORF3 identified by us using ExPASy
Results show that the ABCE1_50 UTR and “no AUG uORFs” constructs express a protein with a molecular weight of 61 kDa, corresponding to the native firefly luciferase (FLuc) protein that is observed in cells transfected with the empty vector

Summary

Introduction

Gene expression is extremely regulated at the level of mRNA translation, especially at its initiation step, where the initiation codon is identified and decoded [1,2]. Translational regulation involves a large number of protein factors and regulatory mRNA sequence elements predominantly located in their 50 and 30 -leader sequences ( known as untranslated regions (UTRs)) [3]. These include small structural elements, internal ribosome entry sites, and regulatory upstream open reading frames (uORFs) [4,5]. With the wide application of ribosome profiling (Ribo-Seq), it was possible to detect a significant ribosomal occupancy in mRNA regions that were thought to be non-coding, including the 50 -leader sequences (i.e., 50 UTRs), which is consistent with the widespread presence of translatable uORFs among the transcriptome [6,7,8]. UORFs are frequently present in transcription factors, cellular receptors, oncogenes, and transcripts with a role in stress response, cell growth, and differentiation control [11,12,13,14]

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