The role of Alternative Polyadenylation on the expression of the alarmin HMGB1.

高迁移率族蛋白1(HMGB1)是一种警报蛋白，由巨噬细胞等免疫细胞分泌，以应对微生物攻击或炎症损伤。虽然HMGB1最初被确定为一种核DNA结合蛋白，其功能是作为某些基因转录的辅助因子，但HMGB1在细胞外环境中的分泌激活了免疫系统中的其他细胞，以分泌额外的促炎细胞因子。有趣的是，细胞分泌的HMGB1水平直接影响其在炎症反应中的功能。事实上，尽管适量的HMGB1可以诱导有益的免疫反应，但过量的HMGB1已被证明是有害的。虽然已知转录调控机制调节HMBG1的表达，但仅靠转录本身并不能解释HMGB1在各种攻击反应中表达的急剧和快速变化。事实上，我们最近证明，转录后机制也在调节HMGB1蛋白水平方面发挥关键作用。我们已经证明，RNA结合蛋白(RBP)HUR通过与HMGB1基因3‘端非翻译区(3’UTR)上的富含U区(称为HuRBS)结合，在肌肉纤维形成过程中起到促进HMGB1翻译的关键作用。我们证明HUR通过抵消microRNA miR-1192的抑制作用来促进HMGB1 mRNA的翻译，miR-1192也被招募到与HuRBS紧邻的种子元件上。令人惊讶的是，尽管这些发现很重要，但我们观察到，突变这种HuRBS并不能完全取消HMGB1的表达，这表明其他元件参与了HMGB1的转录后调控。近年来，交替多聚腺苷酸化(APA)作为一种新的转录后机制出现，它通过改变具有不同长度的3‘UTRs交替多聚腺苷酸化信息中miRNA结合位点的可用性来调节基因表达。由于HMGB1的3‘端非编码区有三个不同的多聚腺苷化位点，即Pas-1、Pas-2和Pas-3，因此HMGB1mRNA可能受到这种机制的调控，这些位点被预测为产生不同长度的mRNA亚型(0.9、1.3和2.4kb)。我们的初步数据表明，HMGB-1的表达确实受到APA介导的机制的调控，因为突变的PAS-2而不是PAS-1或PAS-3会影响小鼠胚胎成纤维细胞中HMGB1的蛋白水平。在准备这一应用时，我们还观察到，尽管2.4和1.3kb(而不是0.89kb)的异构体在非激活的巨噬细胞中表达，但在激活的巨噬细胞中只表达2.4kb的异构体。有趣的是，HMGB1蛋白在这两种情况下仍保持表达。这些观察清楚地表明，根据生长条件的不同，巨噬细胞通过使用1.3kb或2.4kb的mRNA亚型来表达HMGB1蛋白来维持高水平的HMGB1蛋白。我们的数据还表明，1.3和2.4kb亚型在巨噬细胞中的差异表达可能依赖于另一种RBP，PTB1。总而言之，我们的数据使我们假设，在正常情况下和炎症期间，APA可能在调节巨噬细胞中HMGB1的表达方面发挥关键作用。因此，在这项提案中，我们将确定APA影响巨噬细胞中HMGB1转录后调控的机制。特别是，我们建议确定：1)PTB1是否以及如何促进PAS-2而不是PAS-3的选择，以促进非活性巨噬细胞中HMGB1的翻译。2)PTB1是否以miRNA依赖的方式抑制2.4kb HMGB1亚型的翻译。

The high mobility group box 1 (HMGB1) protein is an alarmin that is secreted by immune cells such as macrophages in response to microbial attacks or inflammatory injuries. Although HMGB1 was originally identified as a nuclear DNA-binding protein that functions as a cofactor for the transcription of certain genes, the secretion of HMGB1 in the extracellular milieu activates other cells in the immune system to secrete additional proinflammatory cytokines. Interestingly, the levels of HMGB1 secreted by cells is known to directly affect its function during an inflammatory response. Indeed, although moderate amounts of HMGB1 induce beneficial immune response, excessive levels have been shown to be detrimental. Although transcriptional regulatory mechanisms are known to modulate HMBG1 expression, transcription alone does not explain the drastic and rapid change in HMGB1 expression seen in response to a variety of assaults. Indeed, we recently demonstrated that posttranscriptional mechanisms also play a key in regulating HMGB1 protein levels. We have shown that the RNA binding protein (RBP) HuR plays a key role in promoting HMGB1 translation during muscle fiber formation by binding to an U-rich region (termed the HuRBS) in the 3’untranslated region (3’UTR) of the HMGB1 mRNA. We demonstrate that HuR promotes the translation of HMGB1 mRNA by negating the inhibitory effects of a microRNA, miR-1192, which is also recruited to a seed element located immediately adjacent to the HuRBS. Surprisingly, despite the importance of these findings, we observed that mutating this HuRBS does not completely abrogate the expression of HMGB1 suggesting the involvement of other elements in the posttranscriptional regulation of HMGB1. Recently, the usage of alternate polyadenylation sites, a mechanism referred to as Alternative Polyadenylation (APA), has emerged as a novel posttranscriptional mechanism that modulates gene expression by altering the availability of miRNA binding sites in alternatively polyadenylated messages with 3’UTRs of varying lengths. The HMGB1 mRNA has been suggested to be regulated by such mechanisms since its 3’UTR possesses three different polyadenylation sites, PAS-1, PAS-2, PAS-3, that are predicted to yield mRNA isoforms with different lengths (0.9, 1.3 and 2.4kb). Our preliminary data indicate that HMGB-1 expression is indeed regulated by APA mediated mechanisms since mutating PAS-2 but not PAS-1 or PAS-3 affects HMGB1 protein levels in mouse embryonic fibroblast cells. While preparing this application we also observed that although the 2.4 and 1.3kb (but not the 0.89kb) mRNA isoforms are expressed in inactive macrophages, only the 2.4kb isoform is expressed in activated macrophages. Interestingly the expression of the HMGB1 protein is nonetheless maintained under both conditions. These observations clearly indicate that depending on the growth conditions, macrophages maintain high levels of HMGB1 protein by using either the 1.3kb or 2.4kb mRNA isoform to express the HMGB1 protein. Our data also suggest that the differential expression of the 1.3 and the 2.4kb isoforms in macrophages is possibly dependent on another RBP, PTB1. Collectively our data therefore lead us to hypothesize that APA may play a key role in regulating the expression of HMGB1 in macrophages under normal conditions and during inflammation. Hence in this proposal we will identify the mechanisms through which APA affects the posttranscriptional regulation of HMGB1 in macrophages. Specially, we propose to determine: 1) Whether and how PTB1 promotes the selection of PAS-2 over PAS-3 to promote HMGB1 translation in inactive macrophages. 2) Whether PTB1 suppresses the translation of the 2.4kb HMGB1 isoform in a miRNA dependent manner.