Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Nucleic Acids Res. 2019 Feb 28;47(4):e24. doi: 10.1093/nar/gky1288.
Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.
由相邻基因（顺式剪接）之间的顺式拼接产生的嵌合 RNA（cis-SAGe）越来越被认为是一种广泛存在的现象。这些嵌合信使 RNA 存在于正常的人类细胞中，也在各种癌症中被检测到。由于缺乏可用于其实验研究的可行系统，目前尚不清楚这组嵌合体形成的机制。在这里，我们开发了一种快速、简单且通用的基于细胞的报告系统，以鉴定 cis-SAGe 的调节因子。该报告系统由四个主要盒组成，可同时测量候选调节剂对 cis-SAGe 和规范剪接的影响。使用这种基于细胞的测定法，我们筛选了 102 种候选因子，这些因子涉及 RNA pol II 切割和终止、延伸、剪接、选择性剪接和 R 环形成。我们发现，两个因子，SRRM1 和 SF3B1，不仅影响 cis-SAGe 嵌合体，而且还以全基因组的方式影响其他类型的嵌合 RNA。该系统可用于研究反式作用因子和顺式作用序列元件和因子，以及筛选小分子抑制剂。
