Department of Life Sciences, University of Siena, 53100, Siena, Italy.
Department of Biological, Geological and Environmental Sciences, University of Bologna, Via Selmi 3, 40126, Bologna, Italy.
BMC Bioinformatics. 2024 Sep 2;25(1):286. doi: 10.1186/s12859-024-05909-0.
SmithRNAs (Small MITochondrial Highly-transcribed RNAs) are a novel class of small RNA molecules that are encoded in the mitochondrial genome and regulate the expression of nuclear transcripts. Initial evidence for their existence came from the Manila clam Ruditapes philippinarum, where they have been described and whose activity has been biologically validated through RNA injection experiments. Current evidence on the existence of these RNAs in other species is based only on small RNA sequencing. As a preliminary step to characterize smithRNAs across different metazoan lineages, a dedicated, unified, analytical workflow is needed.
We propose a novel workflow specifically designed for smithRNAs. Sequence data (from small RNA sequencing) uniquely mapping to the mitochondrial genome are clustered into putative smithRNAs and prefiltered based on their abundance, presence in replicate libraries and 5' and 3' transcription boundary conservation. The surviving sequences are subsequently compared to the untranslated regions of nuclear transcripts based on seed pairing, overall match and thermodynamic stability to identify possible targets. Ample collateral information and graphics are produced to help characterize these molecules in the species of choice and guide the operator through the analysis. The workflow was tested on the original Manila clam data. Under basic settings, the results of the original study are largely replicated. The effect of additional parameter customization (clustering threshold, stringency, minimum number of replicates, seed matching) was further evaluated.
The study of smithRNAs is still in its infancy and no dedicated analytical workflow is currently available. At its core, the SmithHunter workflow builds over the bioinformatic procedure originally applied to identify candidate smithRNAs in the Manila clam. In fact, this is currently the only evidence for smithRNAs that has been biologically validated and, therefore, the elective starting point for characterizing smithRNAs in other species. The original analysis was readapted using current software implementations and some minor issues were solved. Moreover, the workflow was improved by allowing the customization of different analytical parameters, mostly focusing on stringency and the possibility of accounting for a minimal level of genetic differentiation among samples.
SmithRNA（小型线粒体高度转录 RNA）是一类新型的小 RNA 分子，它们在线粒体基因组中被编码，并调节核转录物的表达。它们存在的最初证据来自菲律宾蛤仔（Ruditapes philippinarum），在那里已经对其进行了描述，并通过 RNA 注射实验对其活性进行了生物学验证。目前关于这些 RNA 在其他物种中存在的证据仅基于小 RNA 测序。作为在不同后生动物谱系中对 smithRNA 进行特征描述的初步步骤，需要一种专门的、统一的分析工作流程。
我们提出了一种专门针对 smithRNA 的新工作流程。唯一映射到线粒体基因组的序列数据（来自小 RNA 测序）被聚类为假定的 smithRNA，并根据其丰度、在重复文库中的存在以及 5' 和 3' 转录边界的保守性进行预过滤。幸存的序列随后根据种子配对、整体匹配和热力学稳定性与核转录物的非翻译区进行比较，以识别可能的靶标。大量的辅助信息和图形被生成，以帮助在所选物种中对这些分子进行特征描述，并指导操作人员进行分析。该工作流程在原始马尼拉蛤数据上进行了测试。在基本设置下，原始研究的结果在很大程度上得到了复制。进一步评估了额外参数定制（聚类阈值、严格性、最小重复次数、种子匹配）的效果。
SmithRNA 的研究仍处于起步阶段，目前尚无专门的分析工作流程。SmithHunter 工作流程的核心是建立在最初应用于识别马尼拉蛤候选 SmithRNA 的生物信息学程序之上。事实上，这是目前唯一经过生物学验证的 SmithRNA 证据，因此是在其他物种中对 SmithRNA 进行特征描述的首选起点。原始分析使用当前的软件实现进行了重新调整，并解决了一些小问题。此外，通过允许定制不同的分析参数，对工作流程进行了改进，主要侧重于严格性和样本之间遗传分化程度的可能性。
