Characterization of factors and mechanisms of starvation-induced control of TOP mRNA translation

分裂细胞的主要任务是合成和维持翻译装置的组分，包括核糖体蛋白、翻译因子和mRNP相关蛋白。由于这一过程消耗了大部分可用的代谢能量，因此它受到有丝分裂和营养信号的严格调控。在哺乳动物细胞中，这种调节发生在转录后水平，并针对编码翻译机制的蛋白质组分的mRNA。这些mRNA含有一个独特的顺式调控序列元件，称为5'末端寡嘧啶（TOP）基序，它是翻译阻遏蛋白Larp 1的结合位点。在静止或饥饿的细胞中，Larp 1以mTORC 1依赖性方式被募集到TOP基序，导致稳定的亚多聚体mRNP的形成。重要的是，该事件是可逆的，并且当代谢条件改善时允许重建正常翻译模式。虽然Larp 1现在已被公认为TOP mRNA调控的一个关键因素，但对TOP mRNP在饥饿条件下的功能状态、其生化组成以及它们如何逃脱降解知之甚少。在该SPP的第一个资助期内，我们已经建立了用于哺乳动物细胞的Grad-seq技术，并表明Grad-seq能够对细胞提取物中的TOP mRNP调控进行全球调查。与之前得出TOP mRNP在饥饿时从翻译机制中退出的结论相反，我们的Grad-seq和核糖体分析结果认为TOP mRNP采用了新的基线翻译模式。这种不寻常的翻译模式没有观察到典型的（非TOP）mRNA，最有可能是由急剧降低的起始速率。在下一个资助期内，我们将基于这些发现，a）对饥饿诱导的TOP-mRNP进行生物化学表征，B）对TOP反应中起作用的因子进行功能表征，并研究在饥饿期间如何实现和维持基线翻译模式，以及c）确定在恢复正常生长条件后如何重建正常翻译模式。

A major task of dividing cells is the synthesis and maintenance of components of the translation apparatus, including ribosomal proteins, translation factors and mRNP associated proteins. As this process consumes a large proportion of available metabolic energy, it is tightly regulated in response to mitogenic and nutritional signals. In mammalian cells, this regulation occurs on the post-transcriptional level and is directed to mRNAs encoding the protein components of the translation machinery. These mRNAs contain a unique regulatory sequence element in cis, termed the 5' Terminal OligoPyrimidine (TOP) motif, which is the binding site for the translational repressor protein Larp1. In quiescent or starving cells, Larp1 is recruited to the TOP motif in an mTORC1 dependent manner, causing the formation of stable sub-polysomal mRNPs. Importantly, this event is reversible and allows re-establishment of the normal translation mode when metabolic conditions improve. While Larp1 is now well-established as one key factor in TOP mRNA regulation, little is known about the functional status of TOP mRNPs under starvation conditions, their biochemical composition and how they escape degradation. During the first funding period of this SPP, we have established the Grad-seq technique for mammalian cells and shown that Grad-seq enables a global investigation of TOP mRNP regulation in cellular extracts. Contrasting previous reports that concluded that TOP mRNPs are withdrawn from the translation machinery upon starvation, our Grad-seq and ribosome profiling results argue that TOP mRNPs adopt a new baseline translation mode. This unusual translation mode is not observed for canonical (non-TOP) mRNAs and is most likely caused by drastically reduced initiation rates. In the next funding period we will build on these findings to a) biochemically characterize the starvation-induced TOP-mRNP, b) functionally characterize factors that act in the TOP response and investigate how the baseline translation mode is achieved and maintained during starvation and c) identify how the normal translation mode is re-established upon return to normal growth conditions.