Distinct Splice Variants Research Articles

Abstract LB300: RNA splicing variants of the novel long non-coding RNA, CyKILR, exhibit distinct possess divergent biological functions in lung cancer

Abstract The cyclin-dependent kinase inhibitor 2A (CDKN2A) gene encodes the tumor suppressors, p16(INK4A) and p14(ARF), and is found inactivated or deleted in non-small cell lung cancer at a high percentage. In this study, a novel, uncharacterized long non-coding RNA (lncRNA) (ENSG00000267053) located on chromosome 19p13.12 was found overexpressed in non-small cell lung cancer (NSCLC) cells with a wild-type and active CDKN2A gene. The expression of this Cyclin-Dependent Kinase Inhibitor 2A-regulated lncRNA (CyKILR) was also associated with the expression of the tumor suppressor, Liver kinase B1 (LKB1), a product of the STK11 gene. Characterization of CyKILR identified distinct RNA splicing variants classified into two groups based on their exon 3 inclusion/exclusion status, CyKILRa (exon 3 included) and CyKILRb (exon 3 excluded). The CDKN2A and STK11 gene products not only regulated the expression of CyKILR mRNA in non-transformed cells, but also the interplay between these genes synergistically regulated inclusion of exon 3 into the mature CyKILR RNA as shown by dramatic loss of exon 3 inclusion when mRNAs from both tumor suppressor genes were downregulated by siRNA. Functionally, CyKILRa predominantly localized to the nucleus, and targeted downregulation of the CyKILRa using antisense RNA oligonucleotides (ASOs) significantly enhanced cellular proliferation, migration, and clonogenic survival in mechanistic studies. In contrast, CyKILRb predominantly localized to the cytoplasm, and downregulation of CyKILRb using siRNA significantly reduced cell proliferation, migration, and clonogenic survival. Additionally, downregulation of CyKILRb, in stark contrast to CyKILRa, reduced tumor growth in vivo. Interrogation of cell signaling pathways by transcriptomics analyses demonstrated that CyKILRa suppressed key mitogenic pathways further demonstrating the tumor suppressive role of this CyKILR variant. On the other hand, CyKILRb induced key survival pathways further corroborating the oncogenic nature of this variant. Taken together, our findings shed light on the complexity of lncRNA expression and the alternative RNA splicing of lncRNAs as well as their roles in cell signaling and tumorigenesis. Citation Format: Xiujie Xie, Patrick H. Macknight, Amy L. Lu, Charles E. Chalfant. RNA splicing variants of the novel long non-coding RNA, CyKILR, exhibit distinct possess divergent biological functions in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB300.

Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology.

The related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) have diverse biological functions in peripheral tissues and the central nervous system. Therefore, these peptides and their three receptors represent potential drug targets for several conditions, including neurological and pain-related disorders. However, very little is known about how these peptides regulate their receptors through processes such as internalization. Therefore, we developed tools to study receptor regulation through the synthesis of fluorescently labeled analogues of PACAP-38, PACAP-27, and VIP using copper-mediated 1,3-dipolar cycloaddition of the Cy5 fluorophore. The functionality of Cy5-labeled peptides at their receptors was confirmed in cAMP accumulation assays. Internalization of the Cy5-labeled peptides was then examined and quantified at two distinct PAC1 receptor splice variants, VPAC1 and VPAC2 receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manner. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC1n, PAC1s, VPAC1, and VPAC2 receptors. Interestingly, the pattern of Cy5-labeled peptide activity differed for cAMP accumulation and internalization, indicating that these peptides differentially stimulate cAMP accumulation and internalization and therefore display biased agonism. This novel insight into PACAP-responsive receptor signaling and internalization may provide a unique avenue for future therapeutic development. The fluorescently labeled PACAP and VIP peptides described herein, which we validated as tools to study receptor internalization, will have utility across a broad range of applications and provide greater insight into this receptor family.

Abstract 3781: Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression

Abstract Mitochondrial shape is integral for its proper function and is maintained by a dynamic balance between the events of fission and fusion. Hence, a disruption in the balance is detrimental and has been associated with multiple pathologies including tumorigenesis. We noticed significant heterogeneity in mitochondrial morphology and function in ovarian cancer, which remains the deadliest gynecologic malignancy to date. We discovered that heterogenous mitochondrial dynamics in ovarian cancer cells were associated with specific transcript variant signatures of the fission protein Drp1 (encoded by the gene DNM1L), the primary GTPase responsible for mitochondrial fission. While several Drp1 splice variants have been reported, few studies have linked expression and potential interplay of splice variants of Drp1 on mitochondrial dynamics and function with pathophysiology especially in ovarian cancer. We used 3’RACE, western blotting and LC-MS/MS proteomics analysis to establish the identity of the major Drp1 splice variants expressed in ovarian cancer. We found ovarian cancer cell lines as well as patient-ascites derived cells, predominantly express two Drp1 variants: a transcript including both exons 16 and 17 (16/17) and a transcript lacking exon 16 (-/17). We also validated our findings in TCGA ovarian cancer specimens by analyzing Drp1 splice variant transcripts following annotation of TCGA raw RNAseq data and Salmon expression analysis. Our TCGA analysis of these variants highlighted significant difference in overall survival of ovarian cancer patients. Samples with high Drp1(-/17) expression were associated with poorer overall survival compared to those predominantly expressing Drp1(16/17). Furthermore, carrying out gene set enrichment analysis (GSEA) on TCGA specimens split by high expression of these two variants showed enrichment of distinct gene expression signatures. Overexpression and splice variant specific siRNA knockdown studies demonstrated that Drp1 variants have unique localization and effects on mitochondrial morphology and function. Furthermore, metabolic profiling and 13C metabolic flux analysis highlighted variant specific alterations in mitochondrial metabolic pathways and the TCA cycle. Drp1(-/17) expression enhanced mitochondrial respiratory function and as previously shown, Drp1(-/17) associated with both mitochondria and microtubules, potentially implying a more regulated fission activity as a consequence of controlled subcellular localization. Additionally, Drp1(-/17) was enriched and associated with quiescent phenotype compared to more proliferative phenotype of Drp1(16/17). Hence, expression of distinct Drp1 splice variants may be a novel mechanism to regulate mitochondrial fission, and integral to ovarian cancer cell plasticity under different selection pressures during tumor progression. Citation Format: Zaineb Javed, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, Nadine Hempel. Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3781.

Human APOER2 Isoforms Have Differential Cleavage Events and Synaptic Properties.

Human apolipoprotein E receptor 2 (APOER2) is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand-binding domains (LBDs) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared with six identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand-binding repeats generated different amounts of CTFs compared with full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation, respectively, in response to APOE peptide compared with APOER2-FL. The differential CTF generation of Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD, which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses, which may be because of a decrease in the total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.SIGNIFICANCE STATEMENT Humans and mice share virtually the same number of protein-coding genes. However, humans have greater complexity of any higher eukaryotic organisms by encoding multiple protein forms through alternative splicing modifications. Alternative splicing allows pre-mRNAs transcribed from genes to be spliced in different arrangements, producing structurally and functionally distinct protein variants that increase proteomic diversity and are particularly prevalent in the human brain. Here, we identified 25 distinct human APOER2 splice variants from the cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events that exclude N-terminal ligand-binding regions of APOER2. We show that some of the APOER2 variants have differential proteolytic properties in response to APOE ligand and exhibit distinct synaptic properties.

Functional diversity of PFKFB3 splice variants in glioblastomas.

Tumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells. Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered. Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

Identification and function of ETH receptor networks in the silkworm Bombyx mori

Insect ecdysis triggering hormones (ETHs) released from endocrine Inka cells act on specific neurons in the central nervous system (CNS) to activate the ecdysis sequence. These primary target neurons express distinct splicing variants of ETH receptor (ETHR-A or ETHR-B). Here, we characterized both ETHR subtypes in the moth Bombyx mori in vitro and mapped spatial and temporal distribution of their expression within the CNS and peripheral organs. In the CNS, we detected non-overlapping expression patterns of each receptor isoform which showed dramatic changes during metamorphosis. Most ETHR-A and a few ETHR-B neurons produce multiple neuropeptides which are downstream signals for the initiation or termination of various phases during the ecdysis sequence. We also described novel roles of different neuropeptides during these processes. Careful examination of peripheral organs revealed ETHRs expression in specific cells of the frontal ganglion (FG), corpora allata (CA), H-organ and Malpighian tubules prior to each ecdysis. These data indicate that PETH and ETH are multifunctional hormones that act via ETHR-A and ETHR-B to control various functions during the entire development—the ecdysis sequence and associated behaviors by the CNS and FG, JH synthesis by the CA, and possible activity of the H-organ and Malpighian tubules.

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1β, Have Differential Transactivation Activities in Arabidopsis.

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1β. In this study, we examined the differences between the molecular functions of HRE1α and HRE1β. We found that HRE1α and HRE1β are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1β, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1β also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1β was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1β might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1β differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.

Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the “hop cassette”, occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated ‘hopless’. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism’s stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood.

AB0064 CHANGES IN THE VASCULAR ENDOTHELIAL GROWTH FACTOR A (VEGFA) SPLICING AXIS IN HUMAN SYNOVIUM ARE RELATED TO INFLAMMATION IN ARTHRITIS

Background:VEGF-A is a key regulator of rheumatoid (RA) and osteoarthritis (OA). During articular inflammation in OA and RA there is increased synovial angiogenesis and upregulation of angiogenic growth factors such as VEGF-A. VEGF-A comprises two splice variant families, VEGF-Axxxa and VEGF-Axxxb (xxx represents the number of amino acids, from 121 to 206), resulting from alternative splice site selection in exon 8. This site selection is controlled by Serine/Arginine Rich Splicing Factor Kinase 1 (SRPK1), which phophorylates Serine/Arginine Rich Splicing Factor 1 (SRSF1), inducing it to translocate to the nucleus. In most normal tissues, VEGF-Axxxb isoforms predominate, with anti-nociceptive and anti-angiogenic functions. I contrast, in pathological conditions such as inflammation and solid tumours, VEGF-Axxxa isoforms predominate, with pro-nociceptive and pro-angiogenic functions. VEGF-A has been proposed as a therapeutic target in RA and OA. To date, there are no published data on the functionally distinct VEGF-A splice variants in either RA or OA.Objectives:To determine the patterns of, and relationships between, VEGF-A, SRPK1, and SRSF1 expression and activation and synovial inflammation in human RA and OA.Methods:The study was approved by the Nottingham Research Ethics Committee 1 (05/Q2403/24) and Derby Research Ethics Committee 1 (11/H0405/2). Tissues were selected from age- and sex-matched cases in the University of Nottingham joint tissue repository. Post-mortem (PM) samples of healthy knee synovium (n=14, no history of arthritis or knee pain in the 12 months prior to death, no significant articular or synovial pathology) and arthroplasty-derived synovium samples from OA (n=35) or RA (n=14) patients were compared. OA samples were selected to represent the variety of inflammation levels, from low to high grade (0-3, Haywood et al., 2003). 8um thick sections were stained for SRSF1, SRPK1, total VEGF-A and VEGFxxxb via immunohistochemistry. Expression was estimated as fractional area, relative staining intensity (VEGF-Axxxb), and SRSF1 activation quantified by the degree of nuclear localisation. Statistical analyses were performed using Kruskal-Wallis followed by Dunn’s tests and Spearman’s rank correlations.Results:SRPK1 expression was similar across all conditions. SRSF1 showed significantly higher expression in the OA tissue compared to PM (H(2)= 11.29, p=0.002; OA median=0.2, IQR(0.15, 0.28); PM median=0.09, IQR(0.07, 0.16)), and significantly higher nuclear localisation (indicating activation) in RA vs. OA, and in both RA and OA vs PM (H(2)=37.65, p<0.0001 RA cf. PM; p=0,007 OA cf. PM; RA median=89, IQR(83, 93); OA median=36.1, IQR(29, 42); PM median=19.8, IQR(14,21)). Nuclear SRSF1 was significantly correlated with inflammation score (r= 0.52, p<0.05). Total VEGF-A expression was significantly increased in RA compared to PM and OA (H(2)=23.3, p<0.001 RA cf. PM; RA median=0.4, IQR(0.37,0.59); PM median=0.18, IQR(0.15,0.2)) and was also correlated with the severity of inflammation (r=0.47 p<0.05). VEGF-Axxxb showed no changed in expression in OA or RA, although VEGF-Axxxb staining intensity was significant higher in RA samples, compared to controls (H(2)=7.2 p=0.02; RA median=2.3(1, 4); PM median=0.9 (0.7, 1.4)).Conclusion:Increased levels of SRSF1 activation, and the association of total VEGF-A expression with inflammation score, support the hypothesis that there is activation of alternative splicing in inflamed synovium in RA and OA. Targeting this pathway could be a novel therapeutic strategy in OA & RA.

Inflammatory Bowel Disease-associated GP2 Autoantibodies Inhibit Mucosal Immune Response to Adherent-invasive Bacteria.

Adherent-invasive Escherichia coli have been suggested to play a pivotal role within the pathophysiology of inflammatory bowel disease (IBD). Autoantibodies against distinct splicing variants of glycoprotein 2 (GP2), an intestinal receptor of the bacterial adhesin FimH, frequently occur in IBD patients. Hence, we aimed to functionally characterize GP2-directed autoantibodies as a putative part of IBD's pathophysiology. Ex vivo, GP2-splicing variant 4 (GP2#4) but not variant 2 was expressed on intestinal M or L cells with elevated expression patterns in IBD patients. The GP2#4 expression was induced in vitro by tumor necrosis factor (TNF)-α. The IBD-associated GP2 autoantibodies inhibited FimH binding to GP2#4 and were decreased in anti-TNFα-treated Crohn's disease patients with ileocolonic disease manifestation. In vivo, mice immunized against GP2 before infection with adherent-invasive bacteria displayed exacerbated intestinal inflammation. In summary, autoimmunity against intestinal expressed GP2#4 results in enhanced attachment of flagellated bacteria to the intestinal epithelium and thereby may drive IBD's pathophysiology.

Passenger Mutations Confound Phenotypes of SARM1-Deficient Mice

The Toll/IL-1R-domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Roles for SARM1 in TLR signaling, viral infection, inflammasome activation, and chemokine and Xaf1 expression have also been described. Much of the evidence for SARM1 function relies on SARM1-deficient mice generated in 129 ESCs and backcrossed to B6. The Sarm1 gene lies in a gene-rich region encompassing Xaf1 and chemokine loci, which remain 129 in sequence. We therefore generated additional knockout strains on the B6 background, confirming the role of SARM1 in axonal degeneration and WNV infection, but not in VSV or LACV infection, or in chemokine or Xaf1 expression. Sequence variation in proapoptotic Xaf1 between B6 and 129 results in coding changes and distinct splice variants, which may account for phenotypes previously attributed to SARM1. Reevaluation of phenotypes in these strains will be critical for understanding the function of SARM1.

Identification of Novel C-Terminally Truncated Estrogen Receptor β Variant Transcripts and Their Distribution in Humans.

The nuclear receptor genes, including estrogen receptor β (ERβ), contain non-conventional internal and terminal exons, and alternative choice of the exons yields multiple mRNA and protein variants with unique structures and functions. However, the genomic structure of the intronic and 3'-downstream regions of the human ERβ gene and the presence of novel ERβ variants with non-conventional sequences have not been re-examined in about 20 years. Therefore, we attempted to re-characterize the structure of the human ERβ gene and identify novel non-conventional exons and distinct splice variants. Rapid amplification of cDNA 3'-end and RT-PCR cloning were used to isolate human ERβ mRNA variants from the testis. The identified cDNA sequences were mapped on the human genome assembly. Expression profiles of the variants were assessed by RT-PCR analysis. We cloned multiple ERβ mRNA variants with novel nucleotide sequences from the testis and identified several alternative splice sites, 3'-elongation of conventional coding exons, and novel terminal exons in the human ERβ gene. The variants encode C-terminally truncated ERβ proteins termed ERβ6, ERβ7, ERβEx. 4L, and ERβEx. 6L. Furthermore, we identified exon 7-defective forms of ERβ2/βcx, ERβ4, ERβ6, and ERβ7. Subsequently, we noted distinct expression patterns of the variants in human peripheral organs and brain subregions. This study clarified complicated genomic organization and splicing patterns of the human ERβ gene that contribute to the distinct heterogeneity of human ERβ mRNAs and proteins.

An Alternative Splice Variant of HIPK2 with Intron Retention Contributes to Cytokinesis.

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

Skeletal Myosin-Binding Protein C Isoforms Differentially Regulate Fast- and Slow-Twitch Skeletal Muscle Function

Skeletal myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein that is encoded by two paralogous genes (slow-type (MYBPC1) and fast-type (MYBPC2)) and may regulate skeletal muscle contractility. We aimed to characterize the expression pattern, regulatory function, and phosphorylation-dependent modulation of this protein in in vitro actomyosin systems of rat slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL). By immunofluorescence imaging, slow-type MyBP-C is expressed in both EDL and SOL, while the fast-type MyBP-C is exclusive to EDL. Interestingly, these MyBP-C isoforms differentially localize within a discrete region of the sarcomere (the C-zone) that is muscle type-dependent. Mass spectrometry demonstrated expression of 3 distinct N-terminal splice variants of slow-type MyBP-C, which were present in both muscle types. When tissue-purified native thin filaments slid over tissue-purified native myosin thick filaments (containing the physiological MyBP-C compliment) in a calcium-dependent manner, only in the C-zone were thin filaments sensitized to calcium and their velocity slowed. Upon proteolysis of MyBP-C's N terminus, these modulatory capacities were attenuated, implicating this domain in MyBP-C's regulatory effects, as confirmed by in vitro motility experiments using recombinant N-terminal fragments of the various MyBP-C variant isoforms. These isoform fragments displayed differential impacts on thin filament sensitization and slowing, which were significantly reduced following PKA phosphorylation. Finally, we developed a “Monte Carlo” simulation that recapitulates thin filament trajectories observed over thick filaments by assuming that the various MyBP-C isoforms all exist within a C-zone and contribute their variant-specific modulatory impact. Thus, fast- and slow-twitch skeletal muscle contractility are differentially regulated by MyBP-C isoforms, and that MyBP-C function itself is, in turn, regulated by phosphorylation.

ALTERATIONS IN THE MRNA EXPRESSION OF D2 DOPAMINE RECEPTOR SPLICE VARIANTS AS POSSIBLE MECHANISM OF PREFRONTAL DYSFUNCTION AFTER EARLY-LIFE IMMUNE CHALLENGE

Various detrimental factors during early life may affect CNS development and increase risk of neuropsychiatric symptoms in later life. Disruption in brain dopaminergic system maturation is believed to be one of the mechanism of different neurodevelopmental disordrers. In this article we review behavioral peculiarities and changes of prefrontal D2 dopamine receptor splice variants (D2S and D2L) expression in rats after chronic experimental increase of proinflammatory cytokine interleukin(IL)-1β during 3rd week of life. Early life IL-1β treatment produce long lasting working memory deficit originating in juvenile adult animals. Elevation of IL-1β during 3rd week of life also affect developmental expression of D2 dopamine receptor mRNAs leading to increased D2S/D2L ratio in the medial prefrontal cortex of adolescent but not adult rats. Early life IL-1β treatment cancelled the learning-induced D2L mRNA downregulation during active avoidance conditioning in adult rats. Thus, dysregulation of expression of distinct D2 dopamine receptor splice variants within medial prefrontal cortex is supposed to be implicated in cognitive decline caused by early life immune challenge.

Tissue- and cell-specific expression of a splice variant in the II-III cytoplasmic loop of Cacna1b.

Presynaptic CaV2.2 (N‐type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV2.2 channels, Cacna1b, contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a‐Cacna1b and ∆e18a‐Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a‐Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a‐Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a‐Cacna1b is enriched in cholecystokinin‐expressing interneurons. Our results provide key information to understand cell‐specific functions of CaV2.2 channels.

CRISPR‐Cas9 Gene Editing Yields a Novel Rat Model of Cardiometabolic Disease

Despite strong evidence that the Metabolic Syndrome (MetS) and its defining features (central obesity, dyslipidemia, hypertension, hyperglycemia) are highly heritable, the genetic etiology is complex and relatively few causative genes are known. For this reason, we employ the Lyon Hypertensive (LH) rat, a well‐characterized polygenic inbred MetS model. Previous genetic and computational approaches identified a novel gene (C17h6orf52) as a putative master regulator of gene expression and oxidative phosphorylation with pleiotropic effects on body weight and blood pressure regulation.In this study, two different frameshift mutations were generated independently by CRISPR‐Cas9 gene editing, producing either a 5 base pair deletion or a 10 base pair insertion in exon 2 of C17h6orf52, and females from the resulting mutant rat strains, designated M1 and M2, respectively, were characterized (Table 1). Herein, mutations in C17h6orf52 are shown to significantly increase blood pressure and serum cholesterol in vivo, despite no significant changes in body composition or body weight in LH‐derived female rats.Either mutation appears to cause modest, but significant or nearly significant, increases in blood pressure on a 0.3% NaCl diet. M1−/− females exhibit significantly increased systolic and diastolic blood pressure, while M2−/− females' systolic and diastolic blood pressure is more moderately elevated. This effect is potentiated when animals are administered a high salt diet. Finally, although neither total nor low‐density lipoprotein (LDL) cholesterol is changed in the M1 female rats with respect to their wildtype control, M2 females develop higher serum cholesterol, as well as significantly higher LDL cholesterol.The differences in phenotype between the M1 and M2 mutations were unexpected. Preliminary data indicates that 5 base pair deletion present in the M1−/− rats results in two distinct splicing variants that may be contributing to the observed phenotype differences. Nevertheless, these data suggest that C17h6orf52 is protective against cardiometabolic risk factors, given that C17h6orf52 mRNA is more highly expressed in MetS‐susceptible LH rats than MetS resistant Lyon Normotensive (LN) rats, and the mutation confers a greater burden of MetS features on an already obese and hypertensive rat. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate an uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of obesity.Support or Funding InformationR01HL089895, R21DK089417, T32GM008629, 14GRNT20410043This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

Insulin secretion from pancreatic β cells is a highly complex and tightly regulated process. Its dysregulation is one characteristic of type 2 diabetes, and thus, an in-depth understanding of the mechanisms controlling insulin secretion is essential for rational therapeutic intervention. G-protein-coupled receptors (GPCRs) have been established as major regulators of insulin exocytosis. Recent studies also suggest the involvement of adhesion GPCRs, a non-prototypical class of GPCRs. Here, we identify latrophilins, which belong to the class of adhesion GPCRs, to be highly expressed in different cell types of pancreatic islets. Invitro and exvivo analyses show that distinct splice variants of the latrophilin LPHN3/ADGRL3 decrease insulin secretion from pancreatic β cells by reducing intracellular cyclic AMP levels via the Gi-mediated pathway. Our data highlight the key role of LPHN3 in modulating insulin secretion and its potential as therapeutic target for type 2 diabetes.

The antioxidant transcription factor Nrf2 modulates the stress response and phenotype of malignant as well as premalignant pancreatic ductal epithelial cells by inducing expression of the ATF3 splicing variant ΔZip2.

Pancreatic ductal adenocarcinoma (PDAC) exhibits one of the worst survival rates of all cancers. While death rates show declining trends in the majority of cancers, PDAC registers rising rates. Based on the recently described crosstalk between TGF-β1 and Nrf2 in the PDAC development, the involvement of ATF3 and its splice variant ΔZip2 in TGF-β1- and Nrf2-driven pancreatic tumorigenesis was investigated. As demonstrated here, PDAC (Panc1, T3M4) cells or premalignant H6c7 pancreatic ductal epithelial cells differentially express ΔZip2- and ATF3, relating to stronger Nrf2 activity seen in Panc1 cells and TGF-ß1 activity in T3M4 or H6c7 cells, respectively. Treatment with the electrophile/oxidative stress inducer tBHQ or the cytostatic drug gemcitabine strongly elevated ΔZip2 expression in a Nrf2-dependent fashion. The differential expression of ATF3 and ΔZip2 in response to Nrf2 and TGF-ß1 relates to differential ATF3-gene promoter usage, giving rise of distinct splice variants. Nrf2-dependent ΔZip2 expression confers resistance against gemcitabine-induced apoptosis, only partially relating to interference with ATF3 and its proapoptotic activity, e.g., through CHOP-expression. In fact, ΔZip2 autonomously activates expression of cIAP anti-apoptotic proteins. Moreover, ΔZip2 favors and ATF3 suppresses growth and clonal expansion of PDAC cells, again partially independent of each other. Using a Panc1 tumor xenograft model in SCID-beige mice, the opposite activities of ATF3 and ΔZip2 on tumor-growth and chemoresistance were verified in vivo. Immunohistochemical analyses confirmed ΔZip2 and Nrf2 coexpression in cancerous and PanIN structures of human PDAC and chronic pancreatitis tissues, respectively, which to some extent was reciprocal to ATF3 expression. It is concluded that depending on selective ATF3-gene promoter usage by Nrf2, the ΔZip2 expression is induced in response to electrophile/oxidative (here through tBHQ) and xenobiotic (here through gemcitabine) stress, providing apoptosis protection and growth advantages to pancreatic ductal epithelial cells. This condition may substantially add to pancreatic carcinogenesis driven by chronic inflammation.