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大量可用的代谢能量，因此受到有丝分裂和营养信号的严格调控。在哺乳动物细胞中，这种调控发生在转录后水平，并直接针对编码翻译机制蛋白质成分的mrna。这些mrna包含一个独特的顺式调节序列元件，称为5'末端寡聚嘧啶（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依赖的方式被募集到TOP基序，导致稳定的亚多体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建正常的翻译模式。虽然Larp1现在被认为是TOP mRNA调控的一个关键因素，但人们对饥饿条件下TOP mRNA的功能状态、它们的生化组成以及它们如何避免降解知之甚少。在SPP的第一个资助期内，我们已经建立了哺乳动物细胞的Grad-seq技术，并表明Grad-seq能够对细胞提取物中的TOP mRNP调控进行全球研究。之前的报道认为TOP mRNPs在饥饿时退出翻译机制，而我们的gradseq和核糖体分析结果表明，TOP mRNPs采用了一种新的基线翻译模式。这种不寻常的翻译模式在规范（非top） mrna中没有观察到，很可能是由起始率急剧降低引起的。在下一个资助期内，我们将在这些发现的基础上：a)对饥饿诱导的TOP- mrnp进行生化表征；b)从功能上表征在TOP响应中起作用的因子，并研究在饥饿期间如何实现和维持基线翻译模式；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 Contrasting previous reports that concluded that TOP mRNPs are withdrawn from the translation machinery upon starvation, our Grad-seq and ribosome profiling results argue that TOP mRNPs adopt a new baseline translation mode.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.