Deciphering the role of Pumilio1 in two new neurological diseases

关于RNA结合蛋白在大脑发育或疾病中的作用知之甚少，但越来越多的证据表明它们与神经系统疾病有关。事实上，我们发现RNA结合蛋白Pum 1对小鼠和人类的神经功能至关重要。Pum 1单倍体不足的小鼠在5周龄时出现共济失调，并在10周龄时显示浦肯野细胞变性。Pum 1基因敲除小鼠的病情更严重：它们出生时的孟德尔比率较低，比野生型更小，癫痫发作早期，小脑变性更严重。然后我们发现，人类RISK 1的突变也会导致两种非常不同的疾病，这与我们在小鼠中观察到的情况相似：一种轻度、成人发作的纯小脑共济失调，发生在携带突变的患者中，该突变可使β 1水平降低约25%，以及一种早发性疾病，可导致几种认知和身体延迟、体型较小、运动不协调，而在携带突变基因的患者中癫痫发作时，其水平降低了约40- 60%。但是，除了使其不太稳定之外，这些特定的突变是如何改变BMP 1的功能的呢？最明显的地方是在2011年的目标。唯一发表的神经元靶点是ATXN 1和E2 F3，它们的丰度在成人发作性共济失调和早发性病例中增加了相似的量（~50%）。共济失调可能是由小脑ATXN 1丰度升高解释的，但更广泛的发育障碍表型必须涉及其他ATXN 1靶点的失调。然而，这里有一个比最初看起来更令人困惑的问题。最温和的突变T1035 S，它降低了25%，是在同源结构域（HD）;的突变，产生严重的，早发性表型，R1139 W是在HD 8，和R1147 W是在这个域之外。那么，为什么R1139 W和R1147 W产生同样严重的表型，而只有前者消除了R1141的阻遏物活性？为什么T1035 S如此温和，当它也消除了T1031的阻遏物活性时？我们认为，较轻的疾病是由靶点失调引起的，而更严重的疾病则是当β 1的水平低于某个点（可能是30-40%）时引起的，因为它的相互作用伴侣要么不能形成正常的复合物，要么复合物迅速分解，导致这些相互作用物的功能丧失（以及下游靶点功能的丧失或获得）。为了验证这两部分假设，我们将：1）绘制小鼠大脑中的Pum 1靶组以及Pum 2的靶组，Pum 2是其同源物（两种蛋白质之间可能存在调控重叠）; 2）鉴定Pum 1蛋白相互作用物，3）研究小鼠中Pum 1和Pum 2之间的串扰。总之，我们最近的发现不仅定义了两种新的神经系统疾病，它们还证明了理解疾病相关蛋白（如PUF家族）的转录后调控可以导致新的候选疾病基因的鉴定。

Little is known about the role of RNA-binding proteins in brain development or disease, but accumulating evidence indicates their involvement in neurological disorders. In fact, we found that the RNA-binding protein Pum1 is crucial for neurological function in both mice and humans. Pum1-haploinsufficient mice develop ataxia at 5 weeks of age and show Purkinje cell degeneration at 10 weeks. Pum1 knockout mice are sicker: they are born at a lower mendelian ratio, are smaller than wild-type, have early seizures, and more severe cerebellar degeneration. We then found that mutations in human PUM1 also cause two very different diseases that parallel what we observed in mice: a mild, adult-onset pure cerebellar ataxia in patients with a mutation that reduces PUM1 levels by ~25%, and an early-onset disorder that causes several cognitive and physical delays, smaller size, motor incoordination, and seizures in patients with PUM1 mutations that reduce its levels ~40-60%. But how do the specific mutations alter PUM1 function, aside from making it less stable? The most obvious place to look is at PUM1 targets. The only published neuronal targets are ATXN1 and E2F3, and their abundance is increased by similar amounts (~50%) in both the adult-onset ataxia and early-onset cases. The ataxia might be explained by elevated abundance of cerebellar ATXN1, but the broader phenotype of the developmental disorder must involve dysregulation of other PUM1 targets. There is, however, more of a puzzle here than is first apparent. The mildest mutation, T1035S, which reduces PUM1 levels by only 25%, is in homology domain (HD); of the mutations that produce the severe, early onset phenotype, R1139W is in HD8, and R1147W is just outside this domain. Why, then, do R1139W and R1147W produce equally severe phenotypes, when only the former abolishes PUM1's repressor activity? And why is T1035S so mild, when it also abolishes PUM1's repressor activity? We propose that the milder disease results from target dysregulation, whereas more severe disease results when levels of PUM1 fall below a certain point (perhaps 30-40%), because its interacting partners either cannot form their normal complexes or the complexes fall apart quickly, causing loss of function of those interactors (and loss or gain of function of downstream targets). To test this two-part hypothesis, we will: 1) map the Pum1 targetome in the mouse brain as well as that of Pum2, its homolog (there may be regulatory overlap between the two proteins); 2) identify PUM1 protein interactors, and 3) study the cross-talk between Pum1 and Pum2 in mice. In sum, our recent discoveries not only define two new neurological diseases, they demonstrate that understanding the post- transcriptional regulation of disease-related proteins, like the PUF family, can lead to the identification of new candidate disease genes.