Analysis of FGF2 5UTR mRNPs in the control of alternative translation initiation site choice

翻译调控是真核生物基因表达调控的重要和关键环节，允许蛋白质合成的时空调控。它既可以影响蛋白质的量，也可以影响合成的异构体(翻译的数量控制与质量控制)。虽然我们正在慢慢地揭开翻译定量调控的原理，但质量控制仍然大部分是谜。早在20世纪80年代就发现，许多替代翻译起始被一些mRNAs用来从单个RNA物种(如FGF2，C-MYC，VEGF)产生不同的蛋白质亚型。某些细胞条件可以触发N末端延伸的蛋白质异构体的翻译，与较小的蛋白质异构体相比，这些异构体表现出明显不同的活性。举例来说，与小蛋白亚型不同，高相对分子质量(HMW)成纤维细胞生长因子2(FGF2)蛋白不是从细胞中分泌出来的，而是转移到细胞核中，在那里它们表现出永生活性。尽管各种FGF2多肽具有重要的临床意义，但目前仍不清楚单个蛋白亚型的产生是如何被控制的。最近的技术进步使我们能够以高度平行的方式检查核糖核蛋白(RNPs)，同时探测多种蛋白质-RNA相互作用。通过进一步开发、调整和应用这些高通量技术到FGF2mRNA(和一组额外的感兴趣的RNA)，我计划以前所未有的细节询问不同细胞条件下的RNP动态。将这些高通量方法与还原论、机械性实验相结合，将使人们能够对FGF2 mRNA上的翻译起始和起始密码子选择的调节有新的见解，这在生物学中是一个确实重要但知之甚少的过程。

The regulation of translation presents an important and critical layer of gene expression control in eukaryotic organisms allowing for spatio-temporal regulation of protein synthesis. It can affect both the amount of protein and the isoform synthesized (quantitative vs. qualitative control of translation). While we are slowly beginning to unravel the principles underlying quantitative regulation of translation, qualitative control still remains mostly enigmatic.Already in the 1980ies it was discovered that alternative translation initiation was employed by a number of mRNAs to produce different protein isoforms from a single RNA species (such as e.g. FGF2, C-MYC, VEGF). Certain cellular conditions can trigger the translation of N-terminally extended protein isoforms which exhibit markedly different activities compared to their smaller counterparts. Exemplarily, in contrast to the small protein isoform, high molecular weight (HMW) fibroblast growth factor 2 (FGF2) proteins are not secreted from the cell but translocate into the nucleus where they exhibit immortalizing activities. Despite the clinical importance of the various FGF2 peptides it remains unknown how production of the individual protein isoforms is controlled. Recent technological advances now allow us to examine ribonucleoproteins (RNPs) in a highly parallel fashion, probing simultaneously for a multitude of protein-RNA interactions. By further developing, adapting and applying these high throughput techniques to FGF2 mRNA (and a panel of additional RNAs of interest) I plan to interrogate RNP dynamics under different cellular conditions in unprecedented detail. Combining these high throughput approaches with reductionist, mechanistic experimentation will allow novel insights into the regulation of translation initiation and start codon choice on FGF2 mRNA, a truly important but yet poorly understood process in biology.