Antic Sanja, Wolfinger Michael T, Skucha Anna, Hosiner Stefanie, Dorner Silke
Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Institute for Theoretical Chemistry, University of Vienna, Vienna, Austria Center for Integrative Bioinformatics Vienna (CIBIV), Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, Vienna, Austria Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Mol Cell Biol. 2015 Jul;35(13):2309-20. doi: 10.1128/MCB.01346-14. Epub 2015 Apr 27.
The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors, including microRNAs (miRNAs). While it is well established that translation and mRNA degradation are tightly coupled, it is still not entirely clear where in the cell mRNA degradation takes place. In this study, we investigated the possibility of mRNA degradation on the ribosome in Drosophila cells. Using polysome profiles and ribosome affinity purification, we could demonstrate the copurification of various deadenylation and decapping factors with ribosome complexes. Also, AGO1 and GW182, two key factors in the miRNA-mediated mRNA degradation pathway, were associated with ribosome complexes. Their copurification was dependent on intact mRNAs, suggesting the association of these factors with the mRNA rather than the ribosome itself. Furthermore, we isolated decapped mRNA degradation intermediates from ribosome complexes and performed high-throughput sequencing analysis. Interestingly, 93% of the decapped mRNA fragments (approximately 12,000) could be detected at the same relative abundance on ribosome complexes and in cell lysates. In summary, our findings strongly indicate the association of the majority of bulk mRNAs as well as mRNAs targeted by miRNAs with the ribosome during their degradation.
mRNA的翻译和降解是基因表达中的两个关键步骤，受到包括微小RNA（miRNA）在内的许多因素的高度调控和靶向作用。虽然翻译和mRNA降解紧密偶联已得到充分证实，但细胞内mRNA降解发生的位置仍不完全清楚。在本研究中，我们探究了果蝇细胞中核糖体上mRNA降解的可能性。利用多聚核糖体图谱和核糖体亲和纯化技术，我们能够证明各种去腺苷酸化和脱帽因子与核糖体复合物共纯化。此外，AGO1和GW182这两个miRNA介导的mRNA降解途径中的关键因子也与核糖体复合物相关联。它们的共纯化依赖于完整的mRNA，这表明这些因子与mRNA而非核糖体本身相关。此外，我们从核糖体复合物中分离出脱帽的mRNA降解中间体，并进行了高通量测序分析。有趣的是，93%的脱帽mRNA片段（约12,000个）在核糖体复合物和细胞裂解物中以相同的相对丰度被检测到。总之，我们的研究结果有力地表明，大多数大量mRNA以及被miRNA靶向的mRNA在降解过程中与核糖体相关联。
