Additional files, gels, images: A Mammalian Genomic Signature Shaped by Single Nucleotide Variants Regulates Transcriptome Integrity and Diversity

Background: Many functional features of mammalian genomic sequences remain poorly defined, especially how sequence motifs and genetic variants within non-coding regions (NCRs) regulate transcriptome integrity and diversity. We have shown that G-tracts unusually positioned between the polypyrimidine tract and 3′ AG repress usage of the AG and are enriched at cryptic splice sites in cancer cells but their broader role across the extensive NCRs of mammalian genomes is unknown.Results: Here, we identify a widely evolved genomic signature, G-tract-AG motifs consisting of guanine tracts closely upstream of AG dinucleotides, which is significantly associated with single-nucleotide variants (SNVs) identified in genome-wide association studies, particularly within NCRs. Approximately 9,000 such G-tracts within human genes are disrupted by variants of the cis-splicing quantitative trait loci in the Genotype-Tissue Expression project. Functionally, G-tracts repress splicing at the adjacent 3′ AG, primarily by stalling the second transesterification step. Disruption of G-tracts by SNVs relieves this repression, enabling splicing and generating novel transcript isoforms. These G-tract-disrupting SNVs are in cis across the majority of protein-coding genes and are among thousands of rare variants causing genetic diseases. Conclusions: G-tract-AG signatures are widespread bipartite motifs with dual functions: G-tracts repress AG usage to safeguard transcriptome integrity, while SNV-induced disruption releases AGs for splicing to promote transcriptome diversity. Our findings provide mechanistic insights into the regulation of transcriptome integrity and diversity by a mammalian genomic signature, particularly for NCR SNVs associated with diverse traits and a new framework for their functional annotation.

研究背景：哺乳动物基因组序列的诸多功能特征至今仍未得到明确阐释，尤其是非编码区域（non-coding regions, NCRs）内的序列基序与遗传变异如何调控转录组的完整性与多样性。此前我们已发现，异常定位于多聚嘧啶束（polypyrimidine tract）与3′端AG二核苷酸之间的G串（G-tracts）会抑制AG二核苷酸的使用，且在癌细胞的隐蔽剪接位点处富集；但这类序列在哺乳动物基因组广阔的非编码区域中所发挥的更广泛作用仍不明确。
研究结果：本研究鉴定出一类广泛演化的基因组特征——定位于AG二核苷酸紧邻上游的鸟嘌呤串组成的G串-AG基序（G-tract-AG motifs）；该基序与全基因组关联研究中鉴定出的单核苷酸变异（single-nucleotide variants, SNVs）显著相关，尤其在非编码区域内。在基因型-组织表达项目（Genotype-Tissue Expression project, GTEx）中，顺式剪接数量性状位点（cis-splicing quantitative trait loci）的变异会破坏人类基因内约9000个此类G串。功能实验表明，G串主要通过阻滞剪接的第二步转酯化反应，抑制相邻3′端AG二核苷酸处的剪接过程。单核苷酸变异对G串的破坏可解除这种抑制作用，使剪接得以进行并产生全新的转录本异构体。这类破坏G串的单核苷酸变异在大多数蛋白质编码基因中呈顺式分布，且属于引发遗传疾病的数千种罕见变异之列。
研究结论：G串-AG特征属于一类广泛存在的双组分基序，兼具双重功能：G串通过抑制AG二核苷酸的使用以维持转录组的完整性；而单核苷酸变异诱导的G串破坏则可释放AG二核苷酸用于剪接，进而促进转录组的多样性。本研究的发现为解析哺乳动物基因组特征对转录组完整性与多样性的调控机制提供了新视角，尤其针对与多种性状相关的非编码区域单核苷酸变异，并为这类变异的功能注释提供了全新的研究框架。