weak_uORF_Arabidopsis

This dataset is for a paper that study how transcript start sites affect uORF analysis. Abstract: Upstream open reading frames (uORFs) are predicted in over half of the genes in many eukaryotes and often reduce translation and mRNA stability. This raises a paradox: why do uORFs remain so prevalent in eukaryotic genomes despite their repressive effects on gene expression? Using Arabidopsis as a model, we show that transcription start site (TSS) selection controls the inclusion and repressive impact of uORFs within the steady-state RNA pool. We find that TSS selection influences uORF inclusion in > 90% of uORF-containing genes and selectively excludes uORFs from many transcripts. Although bioinformatic analyses predict uORFs in 55% of Arabidopsis genes, fewer than 10% of expressed transcripts contain them. This TSS filtering is nonrandom: genes whose uORFs are excluded are enriched for housekeeping functions, whereas genes retaining uORFs are associated with regulatory roles. We show that failing to account for TSS heterogeneity can mask length-dependent uORF repression and lead to misinterpretation of uORF activities. Importantly, uORFs mostly excluded from native mRNAs can still repress translation when artificially placed upstream of a reporter, demonstrating that apparent uORF repressiveness may not reflect their activity in native transcripts. Our findings establish TSS selection as a major determinant of uORF-mediated translational control. Because TSS heterogeneity is widespread among plant and animal genes, we warn that ignoring TSS heterogeneity can confound analyses of regulatory elements in mRNA 5′ leaders. We therefore recommend accounting for TSS usage when studying uORFs and other mRNA regulatory elements; otherwise, conclusions may reflect regulatory potential rather than regulatory activity in native transcripts.