The human UDP-glycosyltransferase (UGT) gene superfamily generates 22 canonical transcripts coding for functional enzymes and also produces nearly 150 variant UGT transcripts through alternative splicing and intergenic splicing. In the present study, our analysis of circRNA databases identified backsplicing events that predicted 85 circRNAs from UGT genes, with 33, 11, and 19 circRNAs from UGT1A, UGT2B4, UGT8, respectively. Most of these UGT circRNAs were reported by one database and had low abundance in cell- or tissue-specific contexts. Using reverse-transcriptase polymerase chain reaction with divergent primers and cDNA samples from human tissues and cell lines, we found 13 circRNAs from four UGT genes: UGT1A (three), UGT2B7 (one), UGT2B10 (one), and UGT8 (eight). Notably, all eight UGT8 circRNAs contain open reading frames that include the canonical start AUG codon and encode variant proteins that all have the common 274–amino acidN-terminal region of wild-type UGT8 protein. We further showed that one UGT8 circRNA (circ_UGT8-1) was broadly expressed in human tissues and cell lines, resistant to RNase R digestion, and predominately present in the cytoplasm. We cloned five UGT8 circRNAs into the Zinc finger with KRAB and SCAN domains 1 vector and transfected them into HEK293T cells. All these vectors produced both circRNAsand linear transcripts with varying circular/linear ratios (0.17–1.14).Western blotting and mass spectrometry assays revealed that only linear transcripts and not circRNAs were translated. In conclusion, our findings of nearly 100 circRNAs greatly expand the complexity and diversity of the UGT transcriptome; however, UGT circRNAs are expressed at a very low level in specific cellular contexts, and their biologic functions remain to be determined.

中文翻译：

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UDP-糖基转移酶 (UGT) 基因的环状 RNA 扩展了 UGT 转录组的复杂性和多样性

人类 UDP-糖基转移酶 ( UGT ) 基因超家族产生 22 个编码功能酶的经典转录本，并通过选择性剪接和基因间剪接产生近 150 个变异 UGT 转录本。在本研究中，我们对 circRNA 数据库的分析确定了反向剪接事件，这些事件预测了来自UGT基因的85 个circRNA，分别来自UGT1A、UGT2B4、UGT8 的 33、11和 19 个 circRNA 。这些 UGT circRNA 中的大多数是由一个数据库报告的，并且在细胞或组织特异性环境中的丰度较低。使用逆转录酶聚合酶链反应与来自人体组织和细胞系的不同引物和 cDNA 样本，我们发现了来自四个UGT基因：UGT1A（三个）、UGT2B7（一个）、UGT2B10（一个）、UGT8（八）。值得注意的是，所有 8 个 UGT8 circRNA 都包含开放阅读框，其中包括规范起始 AUG 密码子并编码变体蛋白，这些变体蛋白都具有野生型 UGT8 蛋白的共同 274 个氨基酸的 N 端区域。我们进一步表明，一种 UGT8 circRNA（circ_UGT8-1）在人体组织和细胞系中广泛表达，对 RNase R 消化具有抗性，并且主要存在于细胞质中。我们用 KRAB 和 SCAN 结构域 1 载体将 5 个 UGT8 circRNA 克隆到锌指中，并将它们转染到 HEK293T 细胞中。所有这些载体都产生了环状/线性比例不同（0.17-1.14）的 circRNA 和线性转录本。Western 印迹和质谱分析表明，只有线性转录本而不是 circRNA 被翻译。综上所述，我们对近 100 个 circRNA 的发现极大地扩展了 UGT 转录组的复杂性和多样性；然而，UGT circRNAs 在特定细胞环境中的表达水平非常低，其生物学功能仍有待确定。