The patterns of alternative TSS usage explain the highly heterogeneous landscape of 5'UTR lengths in eukaryotes.

Transcription start site (TSS) marks the first DNA nucleotide of a gene transcribed into RNA. Accumulating evidence suggests that alternative TSS usage (ATU) is widespread across eukaryotes, in response to environmental changes and tissue-specific needs. However, how ATU is coordinated with changes in gene transcription activity, and whether it represents a regulated process or merely transcription initiation errors, remains unclear. To address these questions, we conducted integrative analyses of high-resolution TSS maps and translation efficiency (TE) data from multiple eukaryotic organisms. Our results reveal that most ATU events co-occur with differential gene expression and that the direction of ATU is largely consistent with changes in transcription levels. These findings suggest that ATU likely works in concert with transcriptional activity to fine-tune protein production, modulating TE and messenger RNA stability through alterations in 5'UTR sequence and structural features. Given its functional importance, the evolution of TSS locations may have been shaped by natural selection, leading to heterogeneous 5'UTR lengths in genes with distinct expression demands. This study offers new insights into the complexity of gene regulation and provides a plausible explanation for the highly variable 5'UTR landscape observed within and among eukaryotic genes.