Expression Of Different Isoforms Research Articles

Alternative isoform expression of key thermogenic genes in human beige adipocytes

BackgroundThe beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes.MethodsWe performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing.ResultsWe find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT.DiscussionThese results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

Alternative splicing and its regulation in the malaria vector Anopheles gambiae

IntroductionAlternative splicing (AS) is a highly conserved mechanism that allows for the expansion of the coding capacity of the genome, through modifications of the way that multiple isoforms are expressed or used to generate different phenotypes. Despite its importance in physiology and disease, genome-wide studies of AS are lacking in most insects, including mosquitoes. Even for model organisms, chromatin associated processes involved in the regulation AS are poorly known.MethodsIn this study, we investigated AS in the mosquito Anopheles gambiae in the context of tissue-specific gene expression and mosquito responses to a Plasmodium falciparum infection, as well as the relationship between patterns of differential isoform expression and usage with chromatin accessibility changes. For this, we combined RNA-seq and ATAC-seq data from A. gambiae midguts and salivary glands, infected and non-infected.ResultsWe report differences between tissues in the expression of 392 isoforms and in the use of 247 isoforms. Secondly, we find a clear and significant association between chromatin accessibility states and tissue-specific patterns of AS. The analysis of differential accessible regions located at splicing sites led to the identification of several motifs resembling the binding sites of Drosophila transcription factors. Finally, the genome-wide analysis of tissue-dependent enhancer activity revealed that approximately 20% of A. gambiae transcriptional enhancers annotate to a differentially expressed or used isoform, and that their activation status is linked to AS differences between tissues.ConclusionThis research elucidates the role of AS in mosquito vector gene expression and identifies regulatory regions potentially involved in AS regulation, which could be important in the development of novel strategies for vector control.

Differential expression of WNT5A long and short isoforms in non-muscle-invasive bladder urothelial carcinoma.

Wnt ligands belong to a family of secreted glycoproteins in which binding to a range of receptors/co-receptors activates several intracellular pathways. WNT5A, a member of the Wnt family, is classified as a non-canonical Wnt whose activation triggers planar cell polarity (PCP) and Ca+2 downstream pathways. Aberrant expression of WNT5A has been shown to play both protective and harmful roles in an array of conditions, such as inflammatory disease and cancer. In the present study, using histological, immunohistochemical, and molecular methods, we investigated the expression of two isoforms of WNT5A, WNT5A-Short (WNT5A-S) and WNT5A-Long (WNT5A-L) in bladder urothelial carcinoma (UC). Three UC cell lines (RT4, J82, and T24), as well as a normal urothelial cell line, and formalin-fixed, paraffin-embedded (FFPE) transurethral resection (TUR) tissue samples from 17 patients diagnosed with UC were included in the study. WNT5A-L was the predominantly expressed isoform in urothelial cells, although WNT5A-S was also detectable. Further, although no statistically significant difference was found between the percentage of WNT5A-S transcripts in low-grade versus high-grade tumors, we did find a difference between the percentage of WNT5A-S transcripts found in non-invasion versus invasion of the lamina propria, subgroups of non-muscle-invasive tumors. In conclusion, both WNT5A-S and WNT5A-L isoforms are expressed in UC, and the percentage of their expression levels suggests that a higher proportion of WNT5A-S transcription may be associated with lamina propria invasion, a process preceding muscle invasion.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Different expression levels of interleukin-36 in asthma phenotypes

Interleukin (IL)-36 family is closely associated with inflammation and consists of IL-36α, IL-36β, IL-36γ, and IL-36Ra. The role of IL-36 in the context of asthma and asthmatic phenotypes is not well characterized. We examined the sputum IL-36 levels in patients with different asthma phenotypes in order to unravel the mechanism of IL-36 in different asthma phenotypes. Our objective was to investigate the induced sputum IL-36α, IL-36β, IL-36γ, and IL-36Ra concentrations in patients with mild asthma, and to analyze the relationship of these markers with lung function and other cytokines in patients with different asthma phenotypes. Induced sputum samples were collected from patients with mild controlled asthma (n = 62, 27 males, age 54.77 ± 15.49) and healthy non-asthmatic controls (n = 16, 10 males, age 54.25 ± 14.60). Inflammatory cell counts in sputum were determined. The concentrations of IL-36 and other cytokines in the sputum supernatant were measured by ELISA and Cytometric Bead Array. This is the first study to report the differential expression of different isoforms of IL-36 in different asthma phenotypes. IL-36α and IL-36β concentrations were significantly higher in the asthma group (P = 0.003 and 0.031), while IL-36Ra concentrations were significantly lower (P < 0.001) compared to healthy non-asthmatic controls. Sputum IL-36α and IL-36β concentrations in the neutrophilic asthma group were significantly higher than those in paucigranulocytic asthma (n = 24) and eosinophilic asthma groups (n = 23). IL-36α and IL-36β showed positive correlation with sputum neutrophils and total cell count (R = 0.689, P < 0.01; R = 0.304, P = 0.008; R = 0.689, P < 0.042; R = 0.253, P = 0.026). In conclusion, IL-36α and IL-36β may contribute to asthma airway inflammation by promoting neutrophil recruitment in airways. Our study provides insights into the inflammatory pathways of neutrophilic asthma and identifies potential therapeutic target.

Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns.

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.

Insight of Oxidative Stress, Ultrastructural Transformations, and Elemental Variations in Rice Seedlings Exposed to Boron Toxicity: Unraveling the Role of Fe-SOD in Boron Tolerance

Rice (Oryza sativa L.) seedlings differing in boron tolerance were grown in hydroponics containing varying boron concentration. (as boric acid) for 8 days. Yoshida Nutrient Solution (Yoshida et al.1976) served as control, whereas 0.5 mM boron (moderately toxic) and 1.5 mM boron (highly toxic). Boron excess (B) caused marked reduction in length, biomass and relative water content of the seedlings, with more reductions in B-sensitive cv. Malaviya-36 as compared with B-tolerant cv. Brown Gora. B-sensitive seedlings showed higher B uptake in roots and shoots compared to the tolerant. Scanning electron microscopy (SEM) revealed ultrastructural damage to the guard cells with excess boron. Energy dispersive X-ray analysis (EDXA) of rice leaves showed decline in concentration of P, S, Ca and Mg in seedlings on B treatment. Increased production of reactive oxygen species O2˙−, H2O2, lipid peroxides, alteration in activity of antioxidative enzymes and increased membrane permeability was observed in B treated seedlings compared to controls. Real time PCR analysis of stress regulatory genes indicated differential expression of SOD isoforms in the two sets of cultivars with B treatment. Interestingly seedlings of B-tolerant cultivar were characterized by higher level of expression of Fe-SOD and its further increased expression on B treatment. Results suggest that B toxicity involves ultrastructural and elemental changes, increased generation of ROS and altered antioxidative enzyme activities in rice seedlings and increased expression Fe-SOD isoform appears to be associated with B tolerance.

Differential Expression of Insulin Growth Factor 1 (IGF-1) Isoforms in Different Types of Endometriosis: Preliminary Results of a Single-Center Study.

Endometriosis is a benign, estrogen-dependent gynecological condition with an uncertain exact pathogenetic mechanism. The aim of this study was to evaluate the potential differential expression of Insulin Growth Factor 1 (IGF-1) isoforms in deeply infiltrating endometriotic (DIE) lesions, in ovarian endometriomas, and in the eutopic endometrium of the same endometriosis patients and to compare their expression with that in the eutopic endometrium of women without endometriosis. A total of 39 patients were included: 28 with endometriosis, of whom 15 had endometriomas only, 7 had DIE nodules only, and 6 had both DIE and endometriomas, and 11 without endometriosis served as controls. We noticed a similar pattern of expression between IGF-1Ea and IGF-1Ec, which differed from that of the IGF-1Eb isoform, possibly implying differential biological actions of different isoforms in DIE subtypes. We observed a tendency of lower expression of IGF-1Ea and IGF-1Ec in endometriomas without DIE compared to endometriomas with concurrent DIE or in DIE nodules. In conclusion, differential expression of IGF-1 isoforms may indicate that DIE with its associated ovarian lesions and simple ovarian endometriosis should be considered as two forms of the disease developing under different molecular pathways.

Alternative Polyadenylation of Malic Enzyme 1 Is Essential for Accelerated Adipogenesis.

Understanding the mechanism of adipogenesis is an important basis for improving meat quality traits of livestock. Alternative polyadenylation (APA) is a vital mechanism to regulate the expression of eukaryotic genes. However, how the individual APA functions in adipogenesis remains elusive. This study was intended to investigate the effect of malic enzyme 1 (ME1) APA on adipogenesis. Here, intracellular lipid droplets were stained using Oil red O. 3' RACE was used to verify APA events of the ME1 gene. Interactions between ME1 3' untranslated region (3' UTR)-APA isoforms and miRNAs, as well as differential expression of isoforms, were examined using dual-luciferase reporter and molecular experiments. The mechanism of ME1 APA on adipogenesis was explored by gain and loss of function assays. In this study, two ME1 isoforms with different 3' UTR lengths were detected during adipogenesis. Moreover, the ME1 isoform with a short 3' UTR was significantly upregulated compared with the one with a long 3' UTR. Mechanistically, only the long ME1 isoform was targeted by miR-153-3p to attenuate adipogenesis, while the short one escaped the regulation of miR-153-3p to accelerate adipogenesis. Our results reveal a novel mechanism of ME1 APA in regulating adipogenesis.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Tracking of Leukemia and Immune Single Cell Phenotypes during Ipilimumab-Based Treatment By Long-Read Sequencing

Activity of ipilimumab in bone marrow-involved AML has been modest compared to extramedullary AML relapse or solid tumors like melanoma. Single cell transcriptomics (scRNA-seq) could provide deeper understanding of these differences, yet leukemia and immune cell phenotypes defined by somatic mutations or differential isoform expression often cannot be resolved with native short-read scRNA-seq data due to insufficient read coverage. To overcome this limitation, we devised a workflow ( nanoranger) that leverages improved accuracy of long-read sequencing (Oxford Nanopore, ONT) and enables targeted read-out of molecular features including single-nucleotide variants, gene fusions, isoforms, or T cell receptor (TCR) sequences from intermediate scRNA-seq cDNA libraries. To determine the performance of nanoranger, we undertook a series of assessments. First, we established its genotyping accuracy based on the ability to recover highly polymorphic TCR sequences. TCRs obtained with Illumina versus ONT from the same VD(J) amplicon library were highly concordant ( r&amp;gt;0.8) in the number of reads and cells per CDR3 (5,653 [ONT] vs. 5,767 [Illumina]). Second, we tested isoform profiling by targeted sequencing of PTPRC which encodes the T cell differentiation markers CD45RA (exon 4 present) and CD45RO (exon 4 absent). Not only did targeted ONT sequencing increase the single cell coverage of PTPRC from 10 to &amp;gt;2,600 cells, but PTPRC exon 4 expression also correlated with CD45RA surface marker expression by CITE-seq ( r=0.45). Third, we benchmarked somatic mutation detection of a homozygous TP53 R248Gmutation in the AML cell line Kasumi-1. Our ONT-based approach increased genotyped cells from 0% (via native scRNA-seq) to 22% ( nanoranger); in &amp;gt;98.8% of these cells, TP53 R248G was detectable. Finally, we searched for optimal nanoranger targets by assessing whole-transcriptome coverage of 5‘ single cell cDNA and found these to be loci at the 5‘ end of highly expressed transcripts shorter than 4 kB. Altogether, nanoranger reliably recovers molecular features from scRNA-seq libraries with genotyping efficiency dictated by inherent characteristics of the underlying cDNA. To better understand determinants of response and resistance of CTLA-4 blockade in AML, we applied nanoranger to bone marrow samples collected in the context of a phase I trial investigating combined decitabine and ipilimumab (NCT02890329). We genotyped 11 genes recurrently mutated in AML across 15,258 single cell profiles from 13 samples (5 responders at screening and best response, 3 non-responders at screening). At screening, somatic mutations were detectable in myeloid progenitor, monocytic and dendritic cells but also in erythroid and megakaryocytic progenitor populations, consistent with detected copy number changes. Further, we clarified AML subclones with integrated analysis of somatic mutations and chromosomal aberrations. In AML1019, we found two separate clones defined by presence or absence of RUNX1 R320* and amp(8p) or amp(21p). In AML8007, we tracked differential therapeutic responses. At screening, 3 somatic mutations ( DNMT3A V296M, TP53 C176S, TP53 R282W) and 3 chromosomal aberrations ( amp(1p), del(3p) and del(5q)) were detected. At remission, somatic mutations and del(5q) persisted, but amp(1q) and del(3p) became undetectable except in LMPP-like cells. In all 5 responders, somatic mutations remained detectable in all cellular compartments, revealing that the cytoreductive effect of decitabine/ipilimumab was not accompanied by leukemic clone eradication. The ability of nanoranger to perform isoform profiling enabled us to resolve immune phenotypes. Following CTLA-4 blockade, the PTPRC isoform CD45RA was downregulated in CD4 + and CD8 + T cells, consistent with induction of T cell differentiation by ipilimumab. As CTLA-4 exists in two isoforms, we tracked alternative splicing of exon 3 and observed lower expression of the membranous isoform in T cells from AML bone marrow compared to circulating or tumor-infiltrating lymphocytes in melanoma, pointing to context-specific isoform expression of CTLA-4. In sum, genotyping with nanoranger substantially augmented scRNA-seq analyses of ipilimumab-based therapy in bone marrow AML. This exemplifies the great potential that long-read sequencing-based single cell studies hold to better define response and resistance mechanisms of novel immunotherapies.

Multiomics Profiling Reveals Higher RNA Isoform Diversity and Potential Oncospecific Protein Isoform Targets in HSPCs of MDS and MDS/MPN Patients

Background: Myelodysplastic Syndromes/Neoplasms (MDS) are characterized by somatic driver mutations in hematopoietic stem/progenitor cells (HSPCs) leading to hypermethylation, disruption of normal hematopoiesis and progression to acute myeloid leukemia ( Cazzola M, N Engl J Med 2020). With exception of a minority of fit patients, who are candidates for treatment with the potentially curative allogenic hematopoietic stem cell transplantation (allo HSCT), most elderly patients remain incurable and receive palliative treatment with hypomethylating agents. Due to the high phenotypic overlap between normal and dysplastic HSPCs, eradication of diseased cells remains an unmet therapeutic need for patients not eligible for allo HSCT. Aim: We applied a multiomics profiling strategy to identify novel oncospecific protein isoforms as potential drug targets in HSPCs of MDS and MDS/MPN patients. We hypotesized that isoforms might be more specific biomarkers and targets than consensus RNA and proteins. Methods: We included 91 individuals with MDS and MDS/MPN at diagnosis or follow-up without disease modifying treatment (n=71), unclear cytopenia (n=20) and healthy controls (n=6). All samples were collected through our Swiss MDS Registry/Biobank Platform between 8/2017 and 11/2021. We classified all patients clinically by WHO 2016 and genetically using the Malcovati criteria ( Malcovati L et al., Blood 2017). Targeted sequencing of 65 established myeloid driver genes from bulk DNA was performed using the Illumina sequencing platform. RNA and proteins were extracted from selected CD34/CD117 HSPC cells. RNA profiling was performed using libraries generated with the SMART-Seq mRNA reagent and proteotypic profiling was done by tandem mass-spectrometry. RNA isoforms were identified using StringTie ( Pertea M et al., Nature Biotechnology 2015) and protein isoforms using a reference database constructed from the identified RNA isoforms. RMATS was used to detect differences in alternative splicing events. Differential expression of RNA isoforms was analyzed using Ballgown (Frazee A et al. Nat Biotechnol 2015 ) and conventional differential gene-expression was performed by DESeq2 ( Love MI et al., Genome Biology 2014). Results: Genetic classification identified relevant clonality in 2/3 of patients with clinically unclear cytopenia after morphological and cytogenetic assessment (6 MDS, 7 CCUS, 7 ICUS) ( A). The distribution of somatic driver mutations in our cohort was in line with published literature (data not shown). Compared to controls, patients with MDS and MDS/MPN showed a higher rate of intron retentions (data not shown), higher transcriptional diversity of RNA isoforms ( B), lower numbers of differentially over-expressed RNA isoforms (lower isoform abundancy) ( C) but higher numbers of differentially over-expressed genes (higher gene-expression abundancy)( D). This apparently controversial result reflects the methodological bias where RNA isoforms are merged on the corresponding gene-locus by conventional gene-expression analysis missing the full range of transcriptional (isoform) diversity. This RNA isoform diversity was not clearly associated with clinical (MDS vs MDS/MPN) or genetic subgroups (TP53, spliceosome or cell-signaling mutations). Due to low sample size, we could only observe a tendency of higher RNA isoform diversity in TP53 mutated and lower diversity in cell-signaling mutated subgroups ( C). The intersection of differentially up-regulated RNA isoforms and expressed protein isoforms revealed five novel oncospecific proteins (L-P in E) as potential candidates for specific drug targeting in HSPCs of MDS and MDS/MPN. These included an oncospecific membrane protein (L) as well as a cell-signaling enzyme (M) present in about 25%-30% of the diseased samples. Conclusions: With our multiomics profiling strategy, we identified a higher RNA isoform diversity in HSPCs of MDS and MDS/MPN patients as well as five potential oncospecific protein isoform targets. These potential targets can be missed by differential gene-expression ( D) and are better discernible by the biologically more relevant differential RNA-isoform analysis ( C). L and M have well-established functions in stem-cell biology, leukemic transformation and drug resistance making them promising candidates for further functional validation as disease-specific drug targets.

Isoform Specific Roles for the Acute Myeloid Leukemia Stem Cell Antigen CD97

CD97 is an adhesion GPCR protein frequently upregulated in AML and required for LSC self-renewal that is alternatively spliced to form three isoforms that are distinct with respect to their extracellular domain. Our analysis of HSCs from MDS patients with low-risk disease revealed overexpression of CD97 isoform 1 (CD97iso1), while analysis of AML samples from the TCGA dataset revealed preferential expression of CD97iso2 (p&amp;lt;0.0001) (Figure1A). As these data suggested potential isoform specific roles of CD97 in myeloid leukemogenesis, we investigated if the different CD97 isoforms support different functional roles by testing the effect of each isoform on growth, differentiation, and clonogenicity of AML blasts. Consistent with our prior studies demonstrating CD97iso1 promotion of self-renewal in normal CD34+ hematopoietic stem/progenitor cells, over expression of CD97iso1 in AML cell lines increased their serial replating ability, while knockout abrogated their clonogenicity. In stark contrast, overexpression of CD97iso2 or CD97iso3 resulted in decreased clonogenicity upon replating. Overexpression of CD97iso2 conferred the greatest growth advantage to AML cells among the CD97 isoforms. Importantly, knockout of CD97iso2 increased cell differentiation and primed the cells towards apoptosis. Supporting CD97iso2's role in cell growth/survival, daunorubicin treatment increased the expression of CD97iso2. Consistent with CD97's role in increasing migration and invasiveness in solid tumors, we performed trans well migration assays using AML cell lines and observed the highest increase in migration in blasts over expressing CD97iso3 (p&amp;lt;0.01). While CD97 is thought to activate several downstream signaling pathways including the MEK-ERK pathway and PI3K/AKT pathway, it is unknown whether the different CD97 isoforms activate unique downstream signaling pathways, and thus we used an unbiased Bio-ID screen to identify the C-terminal interacting partners of the different CD97 isoforms. We observed preferential enrichment of NLRX1 interactions in CD97iso1 expressing cells, while PI4KA was enriched in CD97iso2 expressing cells. Consistent with NLRX1's role in increasing autophagy, we observed an increase in autophagy upon overexpressing CD97iso1, and consistent with CD97iso2's association with PI4KA, CD97iso2 overexpressing cells showed increased PI4KA and phosphoERK1/2. Finally, confirming non-redundant functional roles of CD97 isoforms in vivo, xenograft studies in NSG mice using AML cell lines revealed that CD97iso2 promoted the most rapid engraftment and shortest survival in recipients (p=0.0001). Given the importance of all the CD97 isoforms in regulating different blast function, we developed antibody-drug conjugates (ADCs) based on human synthetic antibodies that target all isoforms of CD97. ADC against CD97 specifically kills AML cells overexpressing CD97, suggesting the potential use of anti-CD97 ADC for AML therapy. Collectively, these studies demonstrate that CD97 isoforms mediate unique biological roles in vitro and in vivo, and that they likely mediate these functions through differential activation of downstream signaling pathways. We speculate that differential expression of the CD97 isoforms in MDS and AML likely reflects the unique features of MDS HSCs (high levels of CD97iso1 enhancing self-renewal in MDS) versus AML LSCs (CD97iso2 promoting proliferation in AML) (Figure 1B). These studies also highlight the importance of isoform-specific gene expression during myeloid leukemogenesis and suggest that explorations of altered isoform expression in MDS/AML will yield novel insights into the molecular mechanisms that promote disease development and progression.

LEF1 isoforms regulate cellular senescence and aging.

The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high-throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. For the purpose of identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Our analysis identified lymphoid enhancer binding factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with idiopathic pulmonary fibrosis, an age-related disease with strong ties to cellular senescence, revealed a stark dysregulation of LEF1. Collectively, our results suggest that LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.

Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse.

Posttranslational regulation of cardiac NaV1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of NaV channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.

Molecular evidence for the involvement of alternative splicing in jejunum epithelial cells in broiler chickens fed dietary supplementation with olive mill wastewater.

There is no doubt that alternative splicing is conserved in chickens and mammals, but evaluating the effects of nutrition on alternative splicing in chickens is crucial in a wide range of fields. Although the olive diet has been extensively studied in human, mouse, and chicken systems, little is known about its impact on chicken alternative splicing systems. Hence, the current study aimed to assess the effect of feeding polyphenol-enriched olive mill wastewater to female broiler chickens via alternative splicing by analyzing high-throughput sequencing raw reads of RNA utilizing genomics and bioinformatics methodologies. It also aimed to look for differences in isoform expression and discover molecular functions and biological processes linked to differentially transcribed genes. The findings of our study revealed that 51 genes involved in isoform switching and alternative splicing events were not used evenly. This is due to the reduced use of ATSS in olive mill wastewater groups compared to control groups. Furthermore, the gene ontology analysis revealed that 25 GO terms were enriched in biological processes, 16 GO terms were enriched in molecular function, and 25 GO terms were enriched in cellular components. Kinase and adenylyltransferase activities were significantly enriched in terms. The molecular analysis presented herein provides valuable insight into the role of phenolics in alternative gene-splicing mechanisms in chickens, demonstrating how an industrial waste product can be repurposed as a feed supplement with a satisfactory outcome.

P53 Dysregulation in Breast Cancer: Insights on Mutations in the TP53 Network and p53 Isoform Expression.

In breast cancer, p53 expression levels are better predictors of outcome and chemotherapy response than TP53 mutation. Several molecular mechanisms that modulate p53 levels and functions, including p53 isoform expression, have been described, and may contribute to deregulated p53 activities and worse cancer outcomes. In this study, TP53 and regulators of the p53 pathway were sequenced by targeted next-generation sequencing in a cohort of 137 invasive ductal carcinomas and associations between the identified sequence variants, and p53 and p53 isoform expression were explored. The results demonstrate significant variability in levels of p53 isoform expression and TP53 variant types among tumours. We have shown that TP53 truncating and missense mutations modulate p53 levels. Further, intronic mutations, particularly polymorphisms in intron 4, which can affect the translation from the internal TP53 promoter, were associated with increased Δ133p53 levels. Differential expression of p53 and p53 isoforms was associated with the enrichment of sequence variants in p53 interactors BRCA1, PALB2, and CHEK2. Taken together, these results underpin the complexity of p53 and p53 isoform regulation. Furthermore, given the growing evidence associating dysregulated levels of p53 isoforms with cancer progression, certain TP53 sequence variants that show strong links to p53 isoform expression may advance the field of prognostic biomarker study in breast cancer.

Alternative splicing shapes the transcriptome complexity in blackgram [Vigna mungo (L.) Hepper

Vigna mungo, a highly consumed crop in the pan-Asian countries, is vulnerable to several biotic and abiotic stresses. Understanding the post-transcriptional gene regulatory cascades, especially alternative splicing (AS), may underpin large-scale genetic improvements to develop stress-resilient varieties. Herein, a transcriptome based approach was undertaken to decipher the genome-wide AS landscape and splicing dynamics in order to establish the intricacies of their functional interactions in various tissues and stresses. RNA sequencing followed by high-throughput computational analyses identified 54,526 AS events involving 15,506 AS genes that generated 57,405 transcripts isoforms. Enrichment analysis revealed their involvement in diverse regulatory functions and demonstrated that transcription factors are splicing-intensive, splice variants of which are expressed differentially across tissues and environmental cues. Increased expression of a splicing regulator NHP2L1/SNU13 was found to co-occur with lower intron retention events. The host transcriptome is significantly impacted by differential isoform expression of 1172 and 765 AS genes that resulted in 1227 (46.8% up and 53.2% downregulated) and 831 (47.5% up and 52.5% downregulated) transcript isoforms under viral pathogenesis and Fe2+ stressed condition, respectively. However, genes experiencing AS operate differently from the differentially expressed genes, suggesting AS is a unique and independent mode of regulatory mechanism. Therefore, it can be inferred that AS mediates a crucial regulatory role across tissues and stressful situations and the results would provide an invaluable resource for future endeavours in V. mungo genomics.

Limits of the thermal tolerance of two lentic crustaceans: The role of lactate dehydrogenase.

Limits of thermal tolerance in animal life is dependent on energy supply. Accordingly, the lowered ATP production capacity in ectotherms at high temperatures, which arises from a mismatch between oxygen supply and demand and the consequent switch from aerobic to anaerobic metabolism, affects the thermal resistance of these animals. The anaerobic ATP production capacity depends on the functional properties of the enzymes that reduce pyruvate. Thus, the present study focused on the role of the lactate dehydrogenase (LDH) of two daphnid species for anaerobic energy production at warm temperatures and the implications for their specific heat tolerances. Daphnia magna showed a higher thermal limit (indicated by immobilization time at 37°C) than Daphnia pulex, and in both species, this limit increased with rising acclimation temperature. In contrast to D. pulex, D. magna accumulated significant amounts of lactate at higher ambient temperatures. The intensity of anaerobic metabolism was also affected by acclimation temperature. Studying the functional enzyme properties revealed altered maximal reaction rates and substrate inhibitions of the LDH suites of the two daphnid species. D. magna LDH showed a significantly lower substrate inhibition than D. pulex LDH. The LDH isoform composition and the temperature-induced changes differed between both species. The detected qualitative modulations of the LDH suites may have resulted from differential isoform expression and different maturation processes. The species-specific LDH characteristics imply a higher anaerobic energy production at warm temperatures in D. magna, which likely contributes to the higher heat tolerance of this species.