The regulation of gene expression relies heavily on both transcriptional and translational control, yet how these two layers are coordinated remains largely unresolved. In this work, we applied Nanopore long-read sequencing together with cap analysis of gene expression (CAGE-seq) to chart the repertoire of 5′ and 3′ untranslated region (UTR) isoforms and transcription start sites (TSSs) in epidermal stem cells, normal keratinocytes, and squamous cell carcinoma cells. In squamous cell carcinomas, we observed that a select group of genes possessing alternative 5′UTR isoforms displayed markedly higher translational efficiency and were particularly enriched in ribosomal proteins and splicing-related factors. By integrating polysome fractionation with CAGE-seq, we investigated two of these genes sharing identical coding sequences and found that variability in TSS usage frequently resulted in switches between 5′ terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motifs, promoting mTORC1-dependent translation. At a genome-wide scale, transcripts with higher translation in squamous cell carcinoma preferentially used 5′TOP and PRTE motifs, had shorter 5′UTRs, and showed reduced RNA secondary structure. Importantly, only the two RPL21 isoforms containing 5′TOP motifs, not the TOP-lacking versions, significantly correlated with patient overall survival in head and neck squamous cell carcinoma. These findings highlight the need for isoform-specific analysis in cancer studies and indicate that switching 5′UTR isoforms offers a straightforward mechanism to adjust protein synthesis rates, tune mTORC1 responsiveness, and regulate the translational potential of mRNAs through their 5′UTR sequence.
