A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Brandon M Trainor,Arnab Ghosh,Natalia Shcherbik,Dimitri G Pestov,Christopher U T Hellen

doi:10.1038/s41598-021-82025-6

Abstract

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.

Highlights

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions
Modifying the 5ʹ-end of mRNA with a 7-methyl guanosine cap structure (m7G-cap) enables it to interact with the cap-binding protein eIF4E, followed by recruitment of the scaffolding factor eIF4G and DEAD-box RNA helicase eIF4A5,7,8. eIF4F-primed mRNA associates with the 43S pre-initiation complex (43S PIC) composed of a 40S ribosomal subunit bound to eIF1, eIF3, eIF2, and initiator methionyl-tRNA ( tRNAiMet), forming a scanning-competent 48S initiation complex (48S IC)4. 48S IC scans mRNA until it recognizes the first AUG triplet in favorable context, and a codon-anticodon interaction is formed[2], leading to weakening of ribosomal association of e IF19,10
We began addressing the enigmatic nature of black beetle virus (BBV)-seq by examining whether this sequence is capable of promoting translation initiation in a cap-independent manner, and whether it functions as an enhancer/regulatory element in conventional cap-dependent translation

Summary

Introduction

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, capindependent translation. These findings bring new insights into CITE-mediated translational control of gene expression. The most important function of 5ʹ-UTRs is the recruitment of multiple translation initiation factors required for ribosomal subunit association at the correctly selected AUG start codon. Other unstructured (linear) elements of 5ʹ-UTRs include upstream ORFs (uORFs) that lead to reduced expression of the downstream primary O RF13,14, while the mammalian Kozak sequence (5ʹ-GCC(A/G)CCAUGG-3ʹ) flanking the initiation codon promotes its recognition by the scanning complex to ensure fidelity of translation initiation[4,15,16]. Higher-order RNA structures include internal ribosome entry sites (IRESs, discussed in detail below)

Methods

Results

Conclusion