Monitoring the 5′UTR landscape reveals isoform switches to drive translational efficiencies in cancer

Abstract

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5′ and 3′ untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5′UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5′ terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5′TOP and PRTE motifs, have generally shorter 5′UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5′TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5′UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5′UTR sequence.

Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase

“…Cumulative evidence from genome-wide analyses and bioinformatic predictions indicate that thousands of genes may undergo ATI events from both in-frame AUG and near-cognate start codons. This enriches the diversity of the cellular proteome by producing extended or truncated protein isoforms, with the potential gain or loss of functional domains in physiological and pathological situations [49][50][51][52][53][54][55][56][57] . Given this complexity, genetic approaches mutating the main start codons to understand the function of specific protein isoforms must consider the potential contribution of ATI events to the protein pool.…”

Current limitations in predicting mRNA translation with deep learning models

“…Currently, the number of mRNAs whose TE is estimated in a typical experiment is ∼ 20 ′ 000, which corresponds roughly to one isoform per gene. Accurate estimation of the TE of individual isoforms could be an important direction of methodological development [35, 36]. However, it is unlikely that many isoforms are simultaneously expressed at a high enough level to be accurately measured in a given cell type, or that sufficiently accurate data can be currently obtained from single cells [37] that express distinct isoforms.…”

The regulatory landscape of 5′ UTRs in translational control during zebrafish embryogenesis

“…Finally, zebrafish switching 5′ UTR variants can display different motif grammar, and DaniO5P indicates that changes to motif composition impacts their translational effect. Interestingly, cancer cells take advantage of TSS switches to expose or mask motifs to promote translation of a cohort of growth-promoting transcripts (Weber et al , 2023). Moreover, two studies have reported 5′ UTR isoform-specific translation by binding of an RBP to one of the 5′ UTR isoforms but not the other (Aeschimann et al , 2017; Popovitchenko et al , 2020).…”