高通量RNA测序(RNA-seq)和从头转录组组装的兴起对我们如何识别和研究非模型生物的光转导级联中的基因产生了革命性的影响。但是,RNA-seq转录组的几乎自动化注释所提供的优势可能同时阻碍了基因发现和新基因功能发现的可能性。例如,基于与已知蛋白质家族的结构域同源性的标准功能注释只能确认组成员资格,不确定新的生化功能的出现。在这项研究中,我们展示了开发一种策略的重要性,该策略可以规避半自动注释的局限性,并将此工作流程应用于光敏性,作为发现非视蛋白光感受器的一种手段。我们假设非视蛋白G蛋白偶联受体(GPCR)蛋白可能在不同于视蛋白的位置具有发色团结合赖氨酸。这里,我们提供了第一个案例研究,描述了基于普通海湾扇贝Argopectenirradians的组织特异性RNA-seq数据的非视蛋白光敏GPCRs(Lamarck,1819).结合序列分析和三维蛋白质建模,我们确定了两种候选蛋白。我们测试了它们的光化学特性,并提供了证据表明这两种蛋白质掺入了11-顺式和/或全反式视黄醛并与光发生光化学反应。基于这个案例研究,我们证明有可能发现新的光敏GPCRs,我们开发了一个工作流程,从RNA-seq组装到发现新的非视蛋白,基于GPCR的光色素。
The rise of high-throughput RNA sequencing (RNA-seq) and de novo transcriptome assembly has had a transformative impact on how we identify and study genes in the phototransduction cascade of non-model organisms. But the advantage provided by the nearly automated annotation of RNA-seq transcriptomes may at the same time hinder the possibility for gene discovery and the discovery of new gene functions. For example, standard functional annotation based on domain homology to known protein families can only confirm group membership, not identify the emergence of new biochemical function. In this study, we show the importance of developing a strategy that circumvents the limitations of semiautomated annotation and apply this workflow to photosensitivity as a means to discover non-opsin photoreceptors. We hypothesize that non-opsin G-protein-coupled receptor (GPCR) proteins may have chromophore-binding lysines in locations that differ from opsin. Here, we provide the first
case study describing non-opsin light-sensitive GPCRs based on tissue-specific RNA-seq data of the common bay scallop Argopecten irradians (Lamarck, 1819). Using a combination of sequence analysis and three-dimensional protein modeling, we identified two candidate proteins. We tested their photochemical properties and provide evidence showing that these two proteins incorporate 11-cis and/or all-trans retinal and react to light photochemically. Based on this
case study, we demonstrate that there is potential for the discovery of new light-sensitive GPCRs, and we have developed a workflow that starts from RNA-seq assemblies to the discovery of new non-opsin, GPCR-based photopigments.