期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：36
DOI：10.1073/pnas.2122170119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Gene regulation is essential for mammalian development, and dysregulation is linked to many cancers. Scores of mammalian genes have been proposed to have hyperconserved 5′ transcript leaders that direct cap-independent translation via structured internal ribosome entry sites (IRESes). However, this model is based on bicistronic reporter assays, notoriously subject to false-positive results. We show that putative IRESes from
Hoxa9 and other mammalian genes generally encode transcriptional promoters and splice sites, and such features can be used to predict their activity in bicistronic reporters. These false-positive IRESes result from genome annotation errors and transcriptome complexity. Our work suggests these genes are expressed by canonical cap-dependent translation and highlights the importance of accurate transcript annotations for studying translational control.
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.
