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

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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.