Deciphering the role of Pumilio1 in two new neurological diseases

人们对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，它们参与了神经疾病。事实上，我们发现RNA结合蛋白Pum1对小鼠和人类的神经功能都是至关重要的。Pum1单倍体缺陷小鼠在5周龄时出现共济失调，10周龄时出现浦肯野细胞变性。Pum1基因敲除小鼠病情更严重：它们出生时孟德尔比率较低，比野生型小，有早期癫痫发作，小脑变性更严重。然后我们发现，人类PUM1的突变也会导致两种非常不同的疾病，这两种疾病与我们在老鼠身上观察到的情况相似：一种是轻微的成人起病的纯性小脑共济失调，其突变使PUM1水平降低约25%；另一种是早发性疾病，导致PUM1突变患者的几种认知和身体延迟、较小的体积、运动不协调以及癫痫发作，PUM1突变使其水平降低约40-60%。但是，除了使PUM1功能不太稳定外，这些特定的突变还会如何改变PUM1的功能呢？最值得关注的地方是PUM1目标。唯一公布的神经元靶点是ATXN1和E2F3，在成人型和早发性共济失调病例中，它们的丰度增加了类似的量(~50%)。共济失调可以通过小脑ATXN1丰度的增加来解释，但这种发育障碍的更广泛的表型肯定涉及其他PUM1靶点的失调。然而，这里有一个比最初明显更多的谜团。最轻微的突变T1035S仅使PUM1水平降低25%，位于同源区域(HD)；在产生严重、早发表型的突变中，R1139W位于HD8，而R1147W正好位于该区域之外。那么，为什么R1139W和R1147W产生同样严重的表型，而只有前者取消了PUM1的S抑制物活性？为什么T1035S如此温和，而它同时也取消了PUM1的S抑制物活性？我们认为，较轻的疾病是由靶标失调引起的，而当PUM1水平低于某个点(可能是30-40%)时，会导致更严重的疾病，因为它的相互作用伙伴要么不能形成正常的复合体，要么复合体迅速解体，导致这些相互作用子的功能丧失(以及下游靶标的功能丧失或获得)。为了验证这一两部分假说，我们将：1)绘制小鼠脑中的Pum1靶点组以及Pum2的同源基因图(这两种蛋白质之间可能存在调控重叠)；2)确定PUM1蛋白的相互作用；以及3)研究Pum1和Pum2在小鼠体内的相互作用。总之，我们最近的发现不仅定义了两种新的神经系统疾病，它们还表明，了解疾病相关蛋白(如PUF家族)的转录后调控可以导致识别新的候选疾病基因。

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.