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Linking Endoplasmic Reticular Stress and Alternative Splicing.

ABSTRACT: RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 mRNA and yet degrades other cellular mRNAs with related motifs. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2, a transcription elongation factor. ELL2 is necessary for immunoglobulin secretion and does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes.

SUBMITTER: Carew NT

PROVIDER: S-EPMC6321306 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Linking Endoplasmic Reticular Stress and Alternative Splicing.

Carew Nolan T NT Nelson Ashley M AM Liang Zhitao Z Smith Sage M SM Milcarek Christine C

International journal of molecular sciences 20181207 12

RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, <i>ELL2</i> (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of <i>snRNA</i>s. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non ...[more]

PMID: 30544499

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Project description:Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment.

Project description:Several biological effects of haem oxygenase (HO)-1, including anti-inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO-1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO-1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)-3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT-15, LOVO and HT-29 cells in serum-free (SF) conditions with increased HO-1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP-induced apoptosis and HO-1 protein expression in human colon cancer cells. CoPP-induced apoptosis of colon cancer cells was prevented by the addition of the pan-caspase inhibitor, Z-VAD-FMK (VAD), and the Casp-3 inhibitor, Z-DEVD-FMK (DEVD). N-Acetyl cysteine inhibited reactive oxygen species-generated H2 O2 -induced cell death with reduced intracellular peroxide production, but did not affect CoPP-induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO-1 expression by those cells, and knockdown of HO-1 protein expression by HO-1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO4 or biliverdin, induced DNA ladders, and cleavage of Casp-3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM- and CoPP-induced apoptosis. Increased GRP78 level and formation of the HO-1/GRP78 complex were detected in CORM- and CoPP-treated human CRC cells. A pro-apoptotic role of HO-1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.

Project description:Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the oxidative pentose-phosphate pathway (OPPP). The OPPP mainly provides NADPH and sugar-phosphate building blocks for anabolic pathways and is present in all eukaryotes. In plant cells, the irreversible part of the OPPP is found in several compartments. Among the isoforms catalyzing the first OPPP step in Arabidopsis, G6PD1 to G6PD4 target plastids (with G6PD1 being also directed to peroxisomes), whereas G6PD5 and G6PD6 operate in the cytosol. We noticed that alternative splice forms G6PD5.4 and G6PD5.5 encode N-terminally extended proteoforms. Compared to G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts accumulated in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms label different subdomains of the endoplasmic reticulum (ER), and analysis of C-terminal roGFP fusions showed that their catalytic domains face the cytosol. In g6pd5-1 g6pd6-2 mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms were both active and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases (catalyzing the second OPPP step), we noticed that isoform PGL2 carries a C-terminal CaaX motif that may be prenylated for membrane attachment. Reporter-PGL2 fusions co-localized with G6PD5.4 in ER subdomains, which was abolished by Cys-to-Ser exchange in the 256CSIL motif. Among the Arabidopsis 6-phosphogluconate dehydrogenases (catalyzing the third OPPP step), S-acylated peptides were detected for all three isoforms in a recent palmitoylome, with dual cytosolic/peroxisomal PGD2 displaying three sites. Co-expression of GFP-PGD2 diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2's presence. Upon pull-down of GFP-G6PD5.4, not only unlabeled PGD2 and PGL2 were enriched, but also enzymes that depend on NADPH provision at the ER, indicative of physical interaction with the OPPP enzymes. When membrane-bound G6PD5.5 and 5.4 variants were co-expressed with KCR1 (ketoacyl-CoA reductase, involved in fatty acid elongation), ATR1 (NADPH:cytochrome-P450 oxidoreductase), or pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as indirectly (via ATR) NADPH-dependent cytochrome P450 enzyme, co-localization in ER subdomains was observed. Thus, alternative splicing of G6PD5 can direct the NADPH-producing OPPP reactions to the cytosolic face of the ER, where they may operate as membrane-bound metabolon to support several important biosynthetic pathways of plant cells.

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Linking Endoplasmic Reticular Stress and Alternative Splicing.

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Linking Endoplasmic Reticular Stress and Alternative Splicing.

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