Table_3_An Integrated Model of Minor Intron Emergence and Conservation.docx

Minor introns constitute <0.5% of the introns in the human genome and have remained an enigma since their discovery. These introns are removed by a distinct splicing complex, the minor spliceosome. Both are ancient, tracing back to the last eukaryotic common ancestor (LECA), which is reflected by minor intron enrichment in specific gene families, such as the mitogen activated-protein kinase kinases, voltage-gated sodium and calcium ion channels, and E2F transcription factors. Most minor introns occur as single introns in genes with predominantly major introns. Due to this organization, minor intron-containing gene (MIG) expression requires the coordinated action of two spliceosomes, which increases the probability of missplicing. Thus, one would expect loss of minor introns via purifying selection. This has resulted in complete minor intron loss in at least nine eukaryotic lineages. However, minor introns are highly conserved in land plants and metazoans, where their importance is underscored by embryonic lethality when the minor spliceosome is inactivated. Conditional inactivation of the minor spliceosome has shown that rapidly dividing progenitor cells are highly sensitive to minor spliceosome loss. Indeed, we found that MIGs were significantly enriched in a screen for genes essential for survival in 341 cycling cell lines. Here, we propose that minor introns inserted randomly into genes in LECA or earlier and were subsequently conserved in genes crucial for cycling cell survival. We hypothesize that the essentiality of MIGs allowed minor introns to endure through the unicellularity of early eukaryotic evolution. Moreover, we identified 59 MIGs that emerged after LECA, and that many of these are essential for cycling cell survival, reinforcing our essentiality model for MIG conservation. This suggests that minor intron emergence is dynamic across eukaryotic evolution, and that minor introns should not be viewed as molecular fossils. We also posit that minor intron splicing was co-opted in multicellular evolution as a regulatory switch for en masse control of MIG expression and the biological processes they regulate. Specifically, this mode of regulation could control cell proliferation and thus body size, an idea supported by domestication syndrome, wherein MIGs are enriched in common candidate animal domestication genes.

次要内含子（minor introns）在人类基因组的全部内含子中占比不足0.5%，自被发现以来便一直是学界悬而未决的谜题。这类内含子由一套独特的剪接复合体——次要剪接体（minor spliceosome）——负责切除。二者均起源古老，可追溯至真核生物最后共同祖先（last eukaryotic common ancestor, LECA），这一点体现在次要内含子在特定基因家族中的富集现象中，例如丝裂原活化蛋白激酶激酶（mitogen activated-protein kinase kinases）、电压门控钠、钙离子通道（voltage-gated sodium and calcium ion channels）以及E2F转录因子（E2F transcription factors）家族。绝大多数次要内含子以单内含子的形式存在于以主要内含子（major introns）为主的基因中。由于这种基因结构特征，携带次要内含子的基因（minor intron-containing gene, MIG）的表达需要两套剪接体协同运作，这会增加错剪接的发生概率。因此，理论上次要内含子应该会通过纯化选择（purifying selection）发生丢失。这一现象已在至少9个真核支系中导致了次要内含子的完全丢失。然而，次要内含子在陆生植物和后生动物（metazoans）中却高度保守，当次要剪接体失活时会引发胚胎致死，这进一步凸显了次要内含子的重要性。对次要剪接体进行条件性失活的实验表明，快速分裂的祖细胞对次要剪接体的缺失高度敏感。事实上，我们在针对341个增殖细胞系的生存必需基因筛选中发现，MIG显著富集。本研究提出，次要内含子于真核生物最后共同祖先（LECA）时期或更早阶段随机插入基因中，并随后在对增殖细胞存活至关重要的基因中得以保留。我们推测，携带次要内含子的基因的必需性，使得次要内含子在早期真核生物演化的单细胞阶段得以存续。此外，我们鉴定出59个在真核生物最后共同祖先（LECA）之后出现的MIG，其中多数对增殖细胞的存活至关重要，这进一步验证了我们关于MIG保守性的必需性模型。这表明次要内含子的产生在真核生物演化过程中始终处于动态变化之中，不应将其视为分子化石（molecular fossils）。我们还提出，在多细胞生物演化过程中，次要内含子剪接被招募为一种调控开关，用于整体调控MIG的表达及其所参与的生物学过程。具体而言，这种调控模式可通过控制细胞增殖进而影响生物体体型，这一假说得到了驯化综合征（domestication syndrome）的支持：MIG在常见的动物驯化候选基因中显著富集。