Characterization of mRNA-protein complexes as modulators of cellular hypoxia response.

缺氧被描述为细胞缺氧，并强烈影响能量消耗过程，如Na-K-ATP酶和mRNA翻译的活性。因此，在缺氧条件下，5 ′帽依赖性mRNA翻译被抑制。然而，mRNA的一个子集通过不同的机制逃避这种抑制。内部核糖体进入位点（IRES）介导5 ′帽非依赖性翻译起始。在缺氧条件下，ER结合的核糖体有利于翻译。某些mRNA和反式作用蛋白的非翻译区（UTR）中的顺式元件介导它们定位于ER，从而在缺氧条件下有效翻译。利用MCF-7细胞系统，我将利用蛋白质占有率分析研究缺氧反应期间基因表达的转录后控制。通过这种方法将鉴定与蛋白质不同占据的RNA基序。相互作用的RNA结合蛋白（RBP）将通过RNA亲和色谱结合质谱分析或基于其已知的RNA结合基序进行鉴定。动态RNA-蛋白质相互作用在低氧反应过程中的基因表达的转录后控制中的功能将在RNA干扰、mRNA稳定性、核糖体分析和体外翻译实验中进行分析。这些分析将深入了解肿瘤细胞逃避限制肿瘤生长的缺氧条件的机制。

Hypoxia is described as cellular lack of oxygen and strongly affects energy consuming processes as the activity of the Na-K-ATPase and mRNA translation. Thus, under hypoxic conditions global, 5´cap-dependent mRNA translation is inhibited. However, a subset of mRNAs escapes this inhibition by different mechanisms. Internal ribosome entry sites (IRESs) mediate the 5´cap-independent initiation of translation. Under hypoxia translation is favored at ER-bound ribosomes. Cis-elements in the untranslated regions (UTRs) of certain mRNAs and trans-acting proteins mediate their localization to the ER and thereby efficient translation under hypoxic conditions. Employing the MCF-7 cell system I will study the post-transcriptional control of gene expression during the hypoxic response utilizing protein occupancy profiling. RNA motifs differentially occupied with proteins will be identified by this method. Interacting RNA binding proteins (RBPs) will either be identified by RNA affinity chromatography in combination with mass spectrometry analysis or based on their known RNA binding motifs. The function of dynamic RNA-protein interactions in post-transcriptional control of gene expression during the hypoxic response will be analyzed in RNA interference, mRNA stability, ribosome profiling and in vitro translation experiments. These analyses will give insight into mechanisms, by which tumor cells escape hypoxic conditions limiting tumor growth.