The unfolded protein response (UPR) is a collection of pathways that

The unfolded protein response (UPR) is a collection of pathways that maintains the protein secretory pathway during the many physiological and pathological conditions that cause stress in the endoplasmic reticulum (ER). additional RIDD focuses on the sumo transcript does not stably associate with the ER membrane but instead relies on an Xbp1-like stem loop and a second UPR mediator Perk for its degradation during stress. Intro The flux of proteins through the secretory pathway varies extensively among cell types and different pathological and physiological conditions. As demand for secreted proteins changes so do the systems within the endoplasmic reticulum (ER) that are responsible for protein folding and processing. ER stress results when build up of unfolded proteins overcomes the folding capacity of the ER. In metazoans this situation is definitely sensed by three main classes of ER transmembrane proteins- Ire1 Perk and Atf6- which collectively mediate the numerous changes in gene manifestation that define the unfolded protein response (UPR) [1] [2]. This response is essential for normal development in mammals and is thought to effect several diseases including diabetes malignancy and neurodegenerative disorders [3]. The UPR offers broad effects on transcription translation and mRNA decay during ER stress. Translational regulation is definitely mediated mainly by Perk which dimerizes during ER stress and is triggered through autophosphorylation [4] [5]. Perk phosphorylates the translation initiation element eIF2α therefore inhibiting cap-dependent translation of most transcripts [6] [7]. However transcripts comprising upstream open reading frames (uORFs) such as the basic-leucine zipper (b-zip) transcription element Atf4 are selectively translated in these conditions and thus their expression raises during ER stress [8]. Ire1 a second mediator of the UPR oligomerizes during stress leading to activation of its cytosolic kinase and endoribonuclease domains [9] [10] [11]. Ire1 specifically cleaves the mRNA encoding X-box binding protein (Xbp1) directly leading to the cytosolic splicing and translation of this b-zip transcription element [12] [13]. Along with Atf4 and Atf6 (a HMN-214 third b-zip transcription element triggered by proteolysis during ER stress [14]) Xbp1 transcriptionally upregulates many genes encoding ER-specific protein folding chaperones and additional proteins that function in the secretory pathway [15] [16]. Ire1 is also necessary for cleavage HMN-214 of many additional mRNAs initiating their degradation through Regulated Ire1 Dependent Decay (RIDD) [17] [18] [19]. Although much is known about the mechanism of Xbp1 splicing the features of mRNAs that determine them as RIDD focuses on have been more elusive. In cells localization to the ER membrane appears to be the major factor in focusing on mRNAs to this pathway; ER-targeting signals are both necessary and adequate for degradation by RIDD [17] [20] and there is a strong correlation between the degree of membrane association of a given mRNA and its degradation by RIDD during ER stress [20]. Conversely cleavage site specificity does not look like important for RIDD focusing on in are enriched for mRNAs encoding secretory proteins and therefore are presumed to be localized to the ER [18] [19] [21]. However RNA localization does not appear to fully account for the specificity of RIDD in these organisms suggesting that there are additional focusing on requirements. These requirements may include specific sequences such as HMN-214 the stem loop constructions that define the cleavage sites in Xbp1 S100A4 and are also enriched in mammalian RIDD focuses on [18] [19] [22]. Interestingly smt3 the homolog of sumo was recognized in microarray experiments like a potential RIDD target [17] despite lacking any HMN-214 recognizable sequence elements that would target it to the ER. This observation led us to hypothesize the sumo transcript may rely on different mechanisms for degradation compared to the majority of RIDD focuses on in flies. Here we demonstrate the mRNA encoding sumo is definitely a non-canonical RIDD target and depends on both an Xbp1-like stem loop structure and Perk for its degradation HMN-214 during ER stress. Results The mRNA encoding sumo is definitely a non-canonical RIDD target We previously observed by microarray the relative amount of the sumo (smt3 CG4494) transcript decreases during ER stress in S2 cells in an Ire1-dependent but Xbp1-self-employed manner [17]. We confirmed this result here by quantitative real-time PCR (qPCR) (Number 1A-B). Depletion of either Ire1 or Xbp1 by RNAi inhibited the upregulation of BiP a major ER chaperone during ER stress (Number 1A). However depletion of Ire1 but not Xbp1 clogged.