Table_6_Widespread Separation of the Polypyrimidine Tract From 3′ AG by G Tracts in Association With Alternative Exons in Metazoa and Plants.xlsx

At the end of introns, the polypyrimidine tract (Py) is often close to the 3′ AG in a consensus (Y)20NCAGgt in humans. Interestingly, we have found that they could also be separated by purine-rich elements including G tracts in thousands of human genes. These regulatory elements between the Py and 3′ AG (REPA) mainly regulate alternative 3′ splice sites (3′ SS) and intron retention. Here we show their widespread distribution and special properties across kingdoms. The purine-rich 3′ SS are found in up to about 60% of the introns among more than 1,000 species/lineages by whole genome analysis, and up to 18% of these introns contain the REPA G-tracts (REPAG) in about 0.6 million of 3′ SS in total. In particular, they are significantly enriched over their 3′ SS and genome backgrounds in metazoa and plants, and highly associated with alternative splicing of genes in diverse functional clusters. Cryptic splice sites harboring such G- and the other purine-triplets tend to be enriched (2–9 folds over the disrupted canonical 3′ SS) and aberrantly used in cancer patients carrying mutations of the SF3B1 or U2AF35, factors critical for branch point (BP) or 3′ AG recognition, respectively. Moreover, the REPAGs are significantly associated with reduced occurrences of BP motifs between the −24 and −4 positions, in particular absent between the −7 and −5 positions in several model organisms examined. The more distant BPs are associated with increased occurrences of alternative splicing in humans and zebrafish. The REPAGs appear to have evolved in a species- or phylum-specific way. Thus, there is widespread separation of the Py and 3′ AG by REPAGs that have evolved differentially. This special 3′ SS arrangement likely contributes to the generation of diverse transcript or protein isoforms in biological functions or diseases through alternative or aberrant splicing.

在人类基因中，内含子末端的多嘧啶束（polypyrimidine tract, Py）通常以保守序列(Y)₂₀NCAGgt紧邻3'端AG剪接位点。有趣的是，我们在数千个人类基因中发现，二者之间还可存在富含嘌呤的调控元件（包括G束）。这类位于Py与3'端AG之间的调控元件（REPA），主要调控可变3'剪接位点（3' splice site, 3'SS）与内含子滞留过程。本研究展示了这类元件在各生物界的广泛分布与特殊特性。通过全基因组分析，在超过1000个物种类群中，富含嘌呤的3'剪接位点可出现在约60%的内含子中；在总计约60万个3'剪接位点里，其中多达18%的内含子包含REPA G束（REPAG）。尤为值得注意的是，这类元件在后生动物与植物的3'剪接位点及全基因组背景中显著富集，且与多种功能簇基因的可变剪接高度相关。携带此类G束及其他嘌呤三联体的隐蔽剪接位点往往呈现富集态势（较破坏的经典3'剪接位点高2-9倍），且在携带SF3B1或U2AF35突变的癌症患者中被异常使用——这两种因子分别是识别分支点（branch point, BP）与3'端AG的关键蛋白。此外，在检测的多个模式生物中，REPAG与-24至-4位区域内分支点基序的出现频率降低显著相关，尤其在-7至-5位区域完全缺失。在人类与斑马鱼中，位置更远的分支点与可变剪接发生频率升高相关。REPAG似乎以物种或门特异性的方式演化。因此，在生物界中，Py与3'端AG之间通过REPAG产生间隔的现象广泛存在，且这类元件存在差异化演化。这种特殊的3'剪接位点排布模式，可能通过可变剪接或异常剪接参与产生多样的转录本或蛋白质异构体，进而影响生物学功能或疾病进程。