Evolutionary analysis across mammals reveals distinct classes of long noncoding RNAs

Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation. We present slncky, a computational lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we develop a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.近年来，转录组测序（transcriptome sequencing）技术的进步推动了跨多个物种的数千种长链非编码RNA（long non-coding RNAs，lncRNAs）的发现。尽管已有多项研究证实部分lncRNAs在多样的生物学过程中发挥关键功能，但绝大多数lncRNAs的功能与作用机制仍未阐明。在转录组测序与lncRNAs功能注释之间，存在两大显著障碍：一是需从头组装转录组中甄别真正的非编码基因，二是需对上百个候选lncRNAs进行优先级排序以开展后续实验验证。本研究推出slncky，一款计算型lncRNA发现工具，可从RNA测序（RNA-sequencing）数据中筛选得到高质量lncRNA集，并进一步利用进化约束（evolutionary constraint）对具备潜在重要功能的lncRNAs进行优先级排序。我们开发的自动化过滤流程可媲美人工注释工作，且相较于已发表的同类计算方法具有更高的灵敏度。此外，我们还构建了灵敏的lncRNA基因座比对流程，并提出了适用于序列与转录本进化分析的新型进化指标（evolutionary metrics）。分析结果显示，进化选择存在多种不同的作用模式，同时揭示了两类值得关注的基因间lncRNAs（intergenic lncRNAs）：一类在RNA序列层面呈现强烈的纯化选择（purifying selection）特征，另一类的进化约束仅存在于转录本的调控过程中，而非转录本序列本身。