Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas 77030, USA.
Mol Cell Biol. 2011 Jan;31(2):328-41. doi: 10.1128/MCB.00943-10. Epub 2010 Nov 15.
Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3'-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3'-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3'-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3'-end formation and found that processing was strongly dependent upon nucleotides located within the 3' stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3' box. Substitution of the actin promoter for the snRNA promoter abolished proper 3'-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3'-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3'-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.
正确的基因表达依赖于一类广泛表达的、富含尿嘧啶的小核 RNA（snRNA），它们由 RNA 聚合酶 II（RNAPII）转录。脊椎动物 snRNA 由一个独特的启动子转录，该启动子对于正确的 3'-末端形成是必需的，新生转录本的切割涉及一组尚未充分了解的蛋白质的活性，称为整合酶复合物。为了研究果蝇 melanogaster 中的 3'-末端形成，我们开发了一种基于细胞的报告器，通过绿色荧光蛋白（GFP）的表达增加来监测 snRNA 的异常 3'-末端形成。我们在果蝇 S2 细胞中使用该报告器来确定 U7 snRNA 3'-末端形成的要求，发现加工强烈依赖于位于 3'茎环内的核苷酸以及可能构成果蝇中与脊椎动物 3'盒等效的序列。用肌动蛋白启动子代替 snRNA 启动子会破坏正确的 3'-末端形成，这证明了果蝇中 snRNA 启动子的保守要求。我们测试了所有果蝇整合酶亚基的要求，发现整合酶 1、4、9 和 11 对于 3'-末端形成是必需的，而整合酶 3 和 10 可能对于加工是可有可无的。耗尽切割和多聚腺苷酸化因子或组蛋白前体 RNA 加工因子不会影响 U7 snRNA 加工效率，这表明整合酶复合物与 mRNA 3'-末端加工机制没有共享成分。最后，携带整合酶 4 或 7 突变的果蝇不能完成发育，并在幼虫阶段积累大量错误加工的 snRNA。
