Evidence for Widespread Presence of Hidden yet Functional Novel and Non-canonical Human Transcripts

Fraction of functional sequence in human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, like transcription, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse-genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. Altogether, these results argue that many unannotated human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions. Overall design: Used a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool for identification of novel functional genomic elements人类基因组中功能性序列（functional sequence）的占比，始终是生物学领域尚未解决的核心问题之一，也是学界激烈争论的焦点议题。尽管已有大量研究将相当比例的人类DNA与转录等多种生化过程建立关联，但经典的功能定义要求序列能够提供对细胞适合度（cellular fitness）或机体适合度（organismal fitness）产生影响的证据，而这类研究恰恰无法提供此类依据。尽管已有多款高通量反向遗传学筛选（reverse-genetics screens）技术被开发出来以应对这一难题，但这类技术仅能作用于已注释的基因组元件，且存在非特异性效应的局限，因此亟需开发更多功能基因组学（functional genomics）研究方法。本研究构建了一种基于慢病毒（lentivirus）的高通量插入诱变策略，将其作为非整倍体细胞（aneuploid cells）中的正向遗传学筛选工具。将该方法应用于人类细胞系的多表型筛选后，研究发现人类基因组中存在大量未被表征的功能性元件，其中至少部分由已知基因及新发现基因的新型外显子（exon）所构成。携带这些外显子的新型转录本（transcripts）可在特定应激条件下被大规模诱导表达，诱导幅度最高可达数千倍，且其中至少部分可作为双顺反子蛋白编码信使RNA（messenger RNA, mRNA）发挥功能。综合来看，上述研究结果表明，大量未被注释的人类转录本——包括那些看似异常剪接产物的转录本——在特定生物学条件下具备生物学相关性。实验整体设计：采用基于慢病毒的高通量插入诱变策略作为正向遗传学筛选工具，以鉴定新型功能性基因组元件。