Hyperconserved genomic sequences have great promise for understanding core biological processes. It has been recently proposed that scores of hyperconserved 5′ untranslated regions (UTRs), also known as transcript leaders (hTLs), encode internal ribosome entry sites (IRESes) that drive cap-independent translation, in part, via interactions with ribosome expansion segments. However, the direct functional significance of such interactions has not yet been definitively demonstrated. We provide evidence that the putative IRESes previously reported in Hox gene hTLs are rarely included in transcript leaders. Instead, these regions function independently as transcriptional promoters. In addition, we find the proposed RNA structure of the putative Hoxa9 IRES is not conserved. Instead, sequences previously shown to be essential for putative IRES activity encode a hyperconserved transcription factor binding site (E-box) that contributes to its promoter activity and is bound by several transcription factors, including USF1 and USF2 . Similar E-box sequences enhance the promoter activities of other putative Hoxa gene IRESes. Moreover, we provide evidence that the vast majority of hTLs with putative IRES activity overlap transcriptional promoters, enhancers, and 3′ splice sites that are most likely responsible for their reported IRES activities. These results argue strongly against recently reported widespread IRES-like activities from hTLs and contradict proposed interactions between ribosomal expansion segment ES9S and putative IRESes. Furthermore, our work underscores the importance of accurate transcript annotations, controls in bicistronic reporter assays, and the power of synthesizing publicly available data from multiple sources.

Upstream open reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that uORF functions are strongly impacted by both their sequences and locations within transcript leaders. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as important as uORF sequences.

Eukaryotic gene expression is tightly regulated during translation of mRNA to protein. Mis‐regulation of translation can lead to aberrant proteins which accumulate in cancers and cause neurodegenerative diseases. Foundational studies on model genes established fundamental roles for mRNA 5′ transcript leader (TL) sequences in controlling ribosome recruitment, scanning, and initiation. TL cis‐regulatory elements and their corresponding trans‐acting factors control cap‐dependent initiation under unstressed conditions. Under stress, cap‐dependent initiation is suppressed, and specific mRNA structures and sequences promote translation of stress‐responsive transcripts to remodel the proteome. In this review, we summarize current knowledge of TL functions in translation initiation. We focus on insights from high‐throughput analyses of ribosome occupancy, mRNA structure, RNA Binding Protein occupancy, and massively parallel reporter assays. These data‐driven approaches, coupled with computational analyses and modeling, have paved the way for a comprehensive understanding of TL functions. Finally, we will discuss areas of future research on the roles of mRNA sequences and structures in translation. This article is categorized under: Translation > Translation Mechanisms RNA Evolution and Genomics > Computational Analyses of RNA RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

Upstream open reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.

Hyperconserved genomic sequences have great promise for understanding core biological processes. It has been recently proposed that scores of hyperconserved transcript leaders (hTLs) encode Internal Ribosome Entry Sites (IRESes) that drive cap-independent translation in part via interactions with ribosome expansion segments. However, the direct functional significance of such interactions has not yet been definitively demonstrated. We provide evidence that the putative IRESes previously reported in Hoxa gene hTLs are not transcribed. Instead, these regions function independently as transcriptional promoters. In addition, we find the proposed RNA structure of the putative Hoxa9 IRES is not conserved. Instead, sequences previously shown to be essential for putative IRES activity encode a hyperconserved transcription factor binding site (E-box) that contributes to its promoter activity by binding to the transcription factors USF1 and USF2. Similar E-box sequences enhance the promoter activities of other putative Hoxa gene IRESes. Moreover, we provide evidence that the vast majority of hTLs with putative IRES activity overlap transcriptional promoters, enhancers, and 3′ splice sites that are most likely responsible for their reported IRES activities. These results argue strongly against recently reported widespread IRES-like activities from hTLs and contradict proposed interactions between ribosomal expansion segment ES9S and putative IRESes. Our work underscores the importance of accurate transcript annotations, controls in bicistronic reporter assays, and the power of synthesizing publicly available data from multiple sources.