Haenni Simon, Sharpe Helen E, Gravato Nobre Maria, Zechner Kerstin, Browne Cathy, Hodgkin Jonathan, Furger André
Genetics Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Nucleic Acids Res. 2009 Nov;37(20):6723-36. doi: 10.1093/nar/gkp744. Epub 2009 Sep 9.
The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3' flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination.
目前用于描述蛋白质表达基因下游RNA聚合酶II（pol II）转录终止的预测机制，无法充分解释在具有操纵子样结构的真核生物中是如何防止过早终止的。在这里，我们通过分析线虫秀丽隐杆线虫中单个蛋白质表达基因末端以及操纵子内基因的转录终止来解决这个问题。通过结合逆转录-聚合酶链反应（RT-PCR）和染色质免疫沉淀（ChIP）分析，我们发现pol II通常会在终止前转录超过聚腺苷酸化（poly(A)）位点1 kb进入线虫基因的3'侧翼区域。我们还表明，pol II在操纵子内的内部poly(A)位点转录后不会终止。我们提供了实验证据，证明随机选择的五个秀丽隐杆线虫操纵子被转录为多顺反子前体mRNA。此外，我们表明，这些多顺反子前体mRNA中位于下游基因的第一个内含子的顺式剪接对其表达至关重要，并且可能在防止pol II过早转录终止中发挥作用。
