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

细胞分裂的一个主要任务是合成和维持翻译装置的组成部分，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很大比例的可用代谢能量，因此它受到严格调控，以响应有丝分裂和营养信号。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成分的mRNAs。这些mRNA在顺式结构中含有一个独特的调控序列元件，称为5‘末端寡嘧啶(TOP)基序，它是翻译抑制蛋白Larp1的结合位点。在静止或饥饿的细胞中，Larp1以mTORC1依赖的方式被招募到顶端基序，导致稳定的亚多聚体mRNPs的形成。重要的是，这一事件是可逆的，并允许在代谢条件改善时重新建立正常的翻译模式。虽然Larp1现在被认为是TOP mRNA调控的关键因子之一，但人们对TOP mRNPs在饥饿条件下的功能状态、它们的生化组成以及它们如何逃脱降解知之甚少。在这项SPP的第一个资助期，我们已经建立了哺乳动物细胞的Grad-SEQ技术，并表明Grad-SEQ能够对细胞提取液中mRNP的顶级调控进行全球研究。与之前关于顶级mRNP在饥饿时从翻译机制中撤出的结论相反，我们的Grad-Seq和核糖体图谱结果认为，顶级mRNP采用了新的基线翻译模式。这种不寻常的翻译模式在规范(非顶端)的mRNAs中没有观察到，很可能是由于起始率急剧下降所致。在下一个资助期，我们将以这些发现为基础，a)对饥饿诱导的top-mRNP进行生化表征，b)从功能上表征在top反应中起作用的因素，并研究饥饿期间如何实现和保持基线翻译模式，以及c)确定在恢复正常生长条件后如何重新建立正常翻译模式。

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 Contrasting previous reports that concluded that TOP mRNPs are withdrawn from the translation machinery upon starvation, our Grad-seq and ribosome profiling results argue that TOP mRNPs adopt a new baseline translation mode.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.