Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA 15213.
Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2122170119. doi: 10.1073/pnas.2122170119. Epub 2022 Aug 29.
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 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 and . Similar E-box sequences enhance the promoter activities of other putative 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.
高度保守的基因组序列在理解核心生物过程方面具有巨大的潜力。最近有人提出，大量高度保守的 5'非翻译区（UTR），也称为转录前导序列（hTLs），编码内部核糖体进入位点（IRES），这些 IRES 通过与核糖体扩展片段相互作用，部分驱动无帽依赖性翻译。然而，这些相互作用的直接功能意义尚未得到明确证实。我们提供的证据表明，先前在 Hox 基因 hTLs 中报道的假定 IRES 很少包含在转录前导序列中。相反，这些区域独立作为转录启动子发挥作用。此外，我们发现先前提出的假定 IRES 的 RNA 结构并不保守。相反，先前证明对假定 IRES 活性至关重要的序列编码一个高度保守的转录因子结合位点（E 盒），该结合位点有助于其启动子活性，并被包括 和 在内的几个转录因子结合。类似的 E 盒序列增强了其他假定 IRES 基因的启动子活性。此外，我们提供的证据表明，具有假定 IRES 活性的绝大多数 hTLs 与转录启动子、增强子和 3'剪接位点重叠，这些启动子、增强子和 3'剪接位点很可能是其报道的 IRES 活性的原因。这些结果强烈反对最近报道的 hTL 中广泛存在的 IRES 样活性，并与核糖体扩展片段 ES9S 和假定 IRES 之间的提议相互作用相矛盾。此外，我们的工作强调了准确的转录本注释、双顺反子报告基因检测中的对照以及综合来自多个来源的公开可用数据的重要性。
