The Nucleolar δ Isoform of Adapter Protein SH2B1 Enhances Morphological Complexity and Function of Cultured Neurons

The adapter protein SH2B1 is recruited to neurotrophin receptors including TrkB, receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1−/- (KO) mice exhibit decreased neurite complexity and length and BDNF-induced expression of synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, β, and γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ localizes primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc, and FosL1.

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

The Emergence of the Metabolic Signaling of the Nucleoredoxin-like Genes during Evolution

The nucleoredoxin-like genes NXNL1 and NXNL2 were identified through the biological activity of rod-derived cone viability factors (RdCVF and RdCVF2), the alternatively spliced variants produced by intron retention, that mediate signaling between rod and cone photoreceptors by stimulating glucose uptake. These therapeutic genes for inherited retinal degenerations also produce by splicing thioredoxin-like proteins that reduce oxidized cysteines in photoreceptor proteins. The first NXNL genes date from the first animal phyla. Intron retention produces an active RdCVF protein in the tentacles of Hydra vulgaris, a species without eyes. A Scallop RdCVF protein is produced by ciliated photoreceptors of the retina and binds its receptor, BSG1. In the lamprey, a descendent of early vertebrates, RdCVF metabolic signaling between rod and cones is fully established. In the mouse, the production of BSG1 by photoreceptors is regulated by cell-specific splicing inhibition. RdCVF signaling predates photoreceptors and evolved through two alternative splicing events.

Caenorhabditis elegans ETR-1/CELF has broad effects on the muscle cell transcriptome, including genes that regulate translation and neuroblast migration

Migration of neuroblasts and neurons from their birthplace is central to the formation of neural circuits and networks. ETR-1 is the Caenorhabditis elegans homolog of the CELF1 (CUGBP, ELAV-like family 1) RNA-processing factor involved in neuromuscular disorders. etr-1 regulates body wall muscle differentiation. Our previous work showed that etr-1 in muscle has a non-autonomous role in neuronal migration, suggesting that ETR-1 is involved in the production of a signal emanating from body wall muscle that controls neuroblast migration and that interacts with Wnt signaling. etr-1 is extensively alternatively-spliced, and we identified the viable etr-1(lq61) mutant, caused by a stop codon in alternatively-spliced exon 8 and only affecting etr-1 isoforms containing exon 8. We took advantage of viable etr-1(lq61) to identify potential RNA targets of ETR-1 in body wall muscle using a combination of fluorescence activated cell sorting (FACS) of body wall muscles from wild-type and etr-1(lq61) and subsequent RNA-seq. This analysis revealed genes whose splicing and transcript levels were controlled by ETR-1 exon 8 isoforms, and represented a broad spectrum of genes involved in muscle differentiation, myofilament lattice structure, and physiology. Genes with transcripts underrepresented in etr-1(lq61) included those involved in ribosome function and translation, similar to potential CELF1 targets identified in chick cardiomyocytes. This suggests that at least some targets of ETR-1 might be conserved in vertebrates, and that ETR-1 might generally stimulate translation in muscles. As proof-of-principle, a functional analysis of a subset of ETR-1 targets revealed genes involved in AQR and PQR neuronal migration. One such gene, lev-11/tropomyosin, requires ETR-1 for alternative splicing, and another, unc-52/perlecan, requires ETR-1 for the production of long isoforms containing 3′ exons. In sum, these studies identified gene targets of ETR-1/CELF1 in muscles, which included genes involved in muscle development and physiology, and genes with novel roles in neuronal migration.

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Understanding the source of METTL3-independent m6A in mRNA

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30—80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3's role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

Phosphoproteomic Analysis Reveals Downstream PKA Effectors of AKAP Cypher/ZASP in the Pathogenesis of Dilated Cardiomyopathy

Background: Dilated cardiomyopathy (DCM) is a major cause of heart failure worldwide. The Z-line protein Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is closely associated with DCM, both clinically and in animal models. Our earlier work revealed Cypher/ZASP as a PKA-anchoring protein (AKAP) that tethers PKA to phosphorylate target substrates. However, the downstream PKA effectors regulated by AKAP Cypher/ZASP and their relevance to DCM remain largely unknown.Methods and Results: For the identification of candidate PKA substrates, global quantitative phosphoproteomics was performed on cardiac tissue from wild-type and Cypher-knockout mice with PKA activation. A total of 216 phosphopeptides were differentially expressed in the Cypher-knockout mice; 31 phosphorylation sites were selected as candidates using the PKA consensus motifs. Bioinformatic analysis indicated that differentially expressed proteins were enriched mostly in cell adhesion and mRNA processing. Furthermore, the phosphorylation of β-catenin Ser675 was verified to be facilitated by Cypher. This phosphorylation promoted the transcriptional activity of β-catenin, and also the proliferative capacity of cardiomyocytes. Immunofluorescence staining demonstrated that Cypher colocalised with β-catenin in the intercalated discs (ICD) and altered the cytoplasmic distribution of β-catenin. Moreover, the phosphorylation of two other PKA substrates, vimentin Ser72 and troponin I Ser23/24, was suppressed by Cypher deletion.Conclusions: Cypher/ZASP plays an essential role in β-catenin activation via Ser675 phosphorylation, which modulates cardiomyocyte proliferation. Additionally, Cypher/ZASP regulates other PKA effectors, such as vimentin Ser72 and troponin I Ser23/24. These findings establish the AKAP Cypher/ZASP as a signalling hub in the progression of DCM.

Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.

Pyruvate Kinase M1 Regulates Butyrate Metabolism in Cancerous Colonocytes

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.