Kramer Susanne
Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Nucleic Acids Res. 2017 Apr 20;45(7):e49. doi: 10.1093/nar/gkw1245.
The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells.
检测正在进行转录或降解的mRNA具有挑战性，因为这两个过程都很快。然而，处于合成或降解状态的mRNA的相对比例会随着mRNA大小的增加而增加，随着mRNA半衰期的缩短而降低。基于这一原理，我利用了一种22200个核苷酸长的、寿命较短的内源性mRNA作为锥虫中mRNA代谢的报告分子。其5΄和3΄末端分别用红色和绿色荧光的Affymetrix®单mRNA FISH探针标记。在所得的荧光图像中，黄色斑点代表完整的mRNA；红色斑点是处于转录或3΄-5΄降解过程中的mRNA，绿色斑点是处于5΄-3΄降解过程中的mRNA。大多数红色斑点位于细胞核内，对转录抑制不敏感，因此可能是转录中间体。大多数绿色斑点位于细胞质中，这证实了锥虫中大部分细胞质降解是5΄-3΄方向的。该系统在转录或翻译受到抑制以及锥虫与5΄-3΄外切核糖核酸酶Xrn1的同源物被RNA干扰耗尽时呈现出预期的变化。该方法不仅可以在细胞水平以及群体/组织水平上监测mRNA代谢的变化，还可以研究mRNA转录和降解途径的亚细胞定位。我证明了该系统适用于哺乳动物细胞。
