mRNA Isoforms Research Articles

Lifelong Reduction in LDL (Low-Density Lipoprotein) Cholesterol due to a Gain-of-Function Mutation in LDLR.

Loss-of-function mutations in the LDL (low-density lipoprotein) receptor gene (LDLR) cause elevated levels of LDL cholesterol and premature cardiovascular disease. To date, a gain-of-function mutation in LDLR with a large effect on LDL cholesterol levels has not been described. Here, we searched for sequence variants in LDLR that have a large effect on LDL cholesterol levels. We analyzed whole-genome sequencing data from 43 202 Icelanders. Single-nucleotide polymorphisms and structural variants including deletions, insertions, and duplications were genotyped using whole-genome sequencing-based data. LDL cholesterol associations were carried out in a sample of >100 000 Icelanders with genetic information (imputed or whole-genome sequencing). Molecular analyses were performed using RNA sequencing and protein expression assays in Epstein-Barr virus-transformed lymphocytes. We discovered a 2.5-kb deletion (del2.5) overlapping the 3' untranslated region of LDLR in 7 heterozygous carriers from a single family. Mean level of LDL cholesterol was 74% lower in del2.5 carriers than in 101 851 noncarriers, a difference of 2.48 mmol/L (96 mg/dL; P=8.4×10-8). Del2.5 results in production of an alternative mRNA isoform with a truncated 3' untranslated region. The truncation leads to a loss of target sites for microRNAs known to repress translation of LDLR. In Epstein-Barr virus-transformed lymphocytes derived from del2.5 carriers, expression of alternative mRNA isoform was 1.84-fold higher than the wild-type isoform (P=0.0013), and there was 1.79-fold higher surface expression of the LDL receptor than in noncarriers (P=0.0086). We did not find a highly penetrant detrimental impact of lifelong very low levels of LDL cholesterol due to del2.5 on health of the carriers. Del2.5 is the first reported gain-of-function mutation in LDLR causing a large reduction in LDL cholesterol. These data point to a role for alternative polyadenylation of LDLR mRNA as a potent regulator of LDL receptor expression in humans.

Perspective in Alternative Splicing Coupled to Nonsense-Mediated mRNA Decay.

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops. Furthermore, we discuss the importance of AS-NMD in the regulation of biological processes, such as cell differentiation. Finally, we analyze interesting recent data on the relevance of AS-NMD to human health, covering its potential roles in cancer and other disorders.

Transcriptome Analysis of Alternative Splicing Events Induced by Arbuscular Mycorrhizal Fungi (Rhizophagus irregularis) in Pea (Pisum sativum L.) Roots.

Alternative splicing (AS), a process that enables formation of different mRNA isoforms due to alternative ways of pre-mRNA processing, is one of the mechanisms for fine-tuning gene expression. Currently, the role of AS in symbioses formed by plants with soil microorganisms is not fully understood. In this work, a comprehensive analysis of the transcriptome of garden pea (Pisum sativum L.) roots in symbiosis with arbuscular mycorrhiza was performed using RNAseq and following bioinformatic analysis. AS profiles of mycorrhizal and control roots were highly similar, intron retention accounting for a large proportion of the observed AS types (67%). Using three different tools (SUPPA2, DRIMSeq and IsoformSwitchAnalyzeR), eight genes with AS events specific for mycorrhizal roots of pea were identified, among which four were annotated as encoding an apoptosis inhibitor protein, a serine/threonine-protein kinase, a dehydrodolichyl diphosphate synthase, and a pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH1. In pea mycorrhizal roots, the isoforms of these four genes with preliminary stop codons leading to a truncated ORFs were up-regulated. Interestingly, two of these four genes demonstrating mycorrhiza-specific AS are related to the process of splicing, thus forming parts of the feedback loops involved in fine-tuning of gene expression during mycorrhization.

Characterization of mRNA isoform diversity in a transgenic model of tau pathology using targeted long‐read sequencing

AbstractBackgroundAn increasing number of studies implicate a role for alternative splicing in the development and neuropathology of Alzheimer’s disease (AD). However, it has been historically challenging to characterise splicing events, due to the limitations of short‐read RNA‐sequencing (RNA‐Seq) for the capture full‐length transcripts critical for transcriptome assembly. In this study, we used Pacific Biosciences long‐read isoform sequencing (Iso‐Seq) to enrich and comprehensively characterise isoform diversity for AD‐associated genes in entorhinal cortex samples from a well‐validated AD transgenic mouse model.MethodWe used Pacific Biosciences Targeted Iso‐Seq to investigate splicing of 20 AD‐associated genes (including TREM2, BIN1, and APOE) in entorhinal cortex of a well‐characterised mouse model of tau pathology, rTg4510. Sequence data was processed using the Iso‐Seq3 pipeline, followed by downstream analysis using other publically available resources and customised scripts. The same samples have additionally been sequenced using whole transcriptome Iso‐Seq and RNA‐Seq as validation.ResultWe obtained deep sequencing coverage of full‐length transcripts for each of the AD genes, revealing complex usage of alternative start sites and splicing events, as well as many novel 5′starts and 3’ends not previously annotated in existing genomic datasets. We identified differential transcript expression and isoform usage between transgenic and wild‐type mice, with highly‐correlated gene expression between Iso‐Seq and short‐read RNA‐seq data.ConclusionThis study highlights the application of long‐read sequencing approaches to assess splicing variation and isoform diversity in AD by selective gene enrichment. Results suggest differential splicing events associated with AD pathology, supporting a role for transcriptomic dysregulation in development of AD. Further work will be undertaken to characterise other AD‐associated genes, and to extend these analyses to human post‐mortem brain samples.

24 Structural variants affecting mRNAs isoforms splice sites associated with marbling in Nellore cattle

Abstract The mRNA identification and associated splice variants affecting meat marbling in Nellore cattle could contribute to a better understanding of the genetic architecture of this complex trait. Longissimus thoracis samples from the 20 most extreme bulls (out of 80 bulls set) for marbling [high (n=10) and low (n=10) groups] trait were used to perform transcriptomic analysis using RNA-Sequencing. The CLC Genomics Workbench software was used to align the fragments of each sample to the bovine reference genome (ARS-UCD1.2) and to perform differential expressed transcript analysis (DE). An average of 37,000 transcripts and 14 mRNA DE (P ≤ 0.001; FC > 2) were detected in the muscle transcriptome. Among them, three transcripts were up-regulated (COL4A2-202, GHRH-201 and ENSBTAT00000070459.1) and 11 down-regulated (HBB-201, HBA-201, RPL14-202, CATHL1-201, CATHL4-201, MAPKAPK2-204, PCBD1-201, RPL30-203, NEURL1-202, PGLYRP1-201 and ENSBTAT00000038384.2) in high marbling group in relation to low group. Structural variants, including Single Nucleotide Variant (SNV), insertion or deletion were also found in the mRNA DE (4 in high and 6 in low marbling groups), causing possible splice site disruption. For high marbling group, in the COL4A2-202 transcript, three SNVs [one guanine (G) located on chromosome 12 at 85,104,867 bp, two cytosine (C), at the positions: 85,119,139 and 85,145,937 bp] were found. From these, one is a non-synonyms mutation [SNV (G) 85,145,937bp], causing an amino acid change (ENSBTAP00000005916.5: p.Glu1088Asp) in Collagen alpha-2(IV) chain protein (COL4A2). Interestingly, animals with low marbling presented also deletions (85,100,930 pb) in this same gene, causing an amino acid change (ENSBTAP00000005916.5:p.Pro113fs)in the COL4A2 protein. Functional enrichment analysis, using the DE mRNAs list, identified significant metabolic pathways (FDR < 0.05), such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis and phenylalanine degradation. The results suggest potential key regulatory genes associated with marbling, an economically important trait for the beef industry and for the consumer. Funding FAPESP (#2009/16118-5, #11/21241-0, #2017/02470-3, #2017/10630–2, #2018/ 20026–8, #2018/11154-2) and CAPES (#001).

Proteomic and Transcriptomic Analysis Identify Spliceosome as a Significant Component of the Molecular Machinery in the Pituitary Tumors Derived from POU1F1- and NR5A1-Cell Lineages.

Background: Pituitary adenomas (PA) are the second most common tumor in the central nervous system and have low counts of mutated genes. Splicing occurs in 95% of the coding RNA. There is scarce information about the spliceosome and mRNA-isoforms in PA, and therefore we carried out proteomic and transcriptomic analysis to identify spliceosome components and mRNA isoforms in PA. Methods: Proteomic profile analysis was carried out by nano-HPLC and mass spectrometry with a quadrupole time-of-flight mass spectrometer. The mRNA isoforms and transcriptomic profiles were carried out by microarray technology. With proteins and mRNA information we carried out Gene Ontology and exon level analysis to identify splicing-related events. Results: Approximately 2000 proteins were identified in pituitary tumors. Spliceosome proteins such as SRSF1, U2AF1 and RBM42 among others were found in PA. These results were validated at mRNA level, which showed up-regulation of spliceosome genes in PA. Spliceosome-related genes segregate and categorize PA tumor subtypes. The PA showed alterations in CDK18 and THY1 mRNA isoforms which could be tumor specific. Conclusions: Spliceosome components are significant constituents of the PA molecular machinery and could be used as molecular markers and therapeutic targets. Splicing-related genes and mRNA-isoforms profiles characterize tumor subtypes.

VEZF1-guanine quadruplex DNA interaction regulates alternative polyadenylation and detyrosinase activity of VASH1.

Vascular endothelial zinc finger 1 (VEZF1) plays important roles in endothelial lineage definition and angiogenesis. Vasohibins 1 and 2 (VASH1 and VASH2) can form heterodimers with small vasohibin-binding protein (SVBP) and were recently shown to regulate angiogenesis by acting as tubulin detyrosinases. Here, we showed that VEZF1 binds directly with DNA guanine quadruplex (G quadruplex, G4) structures in vitro and in cells, which modulates the levels of the two isoforms of VASH1 mRNA. Disruption of this interaction, through genetic depletion of VEZF1 or treatment of cells with G4-stabilizing small molecules, led to increased production of the long over short isoform of VASH1 (i.e. VASH1A and VASH1B, respectively) mRNA and elevated tubulin detyrosinase activity in cells. Moreover, disruption of VEZF1-G4 interactions in human umbilical vein endothelial cells resulted in diminished angiogenesis. These results suggest that the interaction between VEZF1 and G4 structures assumes a crucial role in angiogenesis, which occurs through regulating the relative levels of the two isoforms of VASH1 mRNA and the detyrosinase activity of the VASH1-SVBP complex. Together, our work revealed VEZF1 as a G4-binding protein, identified a novel regulatory mechanism for tubulin detyrosinase, and illustrated that the VEZF1- and VASH1-mediated angiogenesis pathways are functionally connected.

Abstract 17448: Comprehensive Characterization of Alternative Polyadenylation in Human Right Ventricle Failure

Background: Alternative cleavage and polyadenylation (APA) an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3’UTR lengths. APA plays an important role in different biological processes and dysregulation of APA leading to many human diseases. However, the functional consequences of APA events in the right ventricle (RV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the RV failure in humans compared to healthy RV. Methods and Results: We used Poly A tail RNA sequencing and a novel algorithm called DaPars to measure the global patterns of APA in healthy and failing human RV specimens. We revealed 3'UTR shortening and lengthening of many genes in failing RV compared to healthy RV specimens. The lengthened 3’UTR genes were enriched for functional groups such as RNA processing and splicing. Whereas the shortened 3’UTR genes were enriched for nucleic acid metabolic process and chromatin organization. In addition, differential APA events in RV failure regulates many pathways important for the progression of the RV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in RV failure compared to healthy RV specimens. Conclusions: Our results highlight important roles of APA in the progression of RV failure in humans, including RNA processing, splicing, and specific gene expression. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in RV failure.

Mechanisms regulating DMTF1β/γ expression and their functional interplay with DMTF1α.

The cyclin D binding myb-like transcription factor 1 (DMTF1), a haplo-insufficient tumor suppressor gene, has 3 alternatively spliced mRNA isoforms encoding DMTF1α, β and γ proteins. Previous studies have indicated a tumor suppressive role of DMTF1α and the oncogenic activity of DMTF1β, while the function of DMTF1γ remains largely undetermined. In the present study, the mechanisms regulating DMTF1 isoform expression were investigated and the functional interplay of DMTF1β and γ with DMTF1α was characterized. It was found that specific regions of DMTF1β and γ transcripts can impair their mRNA integrity or stability, and thus reduce protein expression levels. Additionally, DMTF1β and γ proteins exhibited a reduced stability compared to DMTF1α and all 3 DMTF1 isoforms were localized in the nuclei. Two basic residues, K52 and R53, in the DMTF1 isoforms determined their nuclear localization. Importantly, both DMTF1β and γ could associate with DMTF1α and antagonize its transactivation of the ARF promoter. Consistently, the ratios of both DMTF1β/α and γ/α were significantly associated with a poor prognoses of breast cancer patients, suggesting oncogenic roles of DMTF1β and γ isoforms in breast cancer development.

Pp90RSK Isoforms Play Distinct Roles during Hematopoiesis

Ribosomal S6 Kinases (RSKs) include a family of serine/threonine kinases that regulate cell proliferation and survival. In humans, four RSK isoforms (RSK1-4) have been identified. Although they are 73-80% conserved in sequence homology, RSK isoforms are reported to have distinct functions. RSKs phosphorylate cyclic AMP response element binding protein (CREB) which is a regulator of hematopoietic proliferation and differentiation. RSK1 is hyperactivated in AML and is therefore a potential therapeutic target for acute myeloid leukemia (AML). Therefore, inhibition of RSK requires understanding of its role during normal hematopoiesis. Furthermore, the role of RSK isoforms during hematopoiesis and leukemogenesis has not been well characterized. To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p<0.05) in the earlier stages of normal hematopoiesis. Moreover, RPS6KA3 is highly expressed in CD38-CD34+ HSCs compared with other progenitor populations (LMPP, MPP, CMP, GMP, and MEP) isolated from human cord blood. RPS6KA2 expression was higher in LMPP by 7-fold (p<0.05) and CMP by 3-fold (p<0.05) compared with HSCs but was barely detectable in MEPs. RPS6KA1 was ubiquitously expressed in all progenitor populations. These results suggest that RSK2 and RSK3 have distinct functions during early myelopoiesis. To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p<0.05) compared with control cells, RSK1 knockdown decreased BFU-E and CFU-E erythroid colonies by 2-fold (p<0.05) but increased CFU-GM colonies by 2-fold (p<0.05) compared with control cells. However, RSK2 knockdown did not show any significant effects on colony-forming activity of HSPCs. We confirmed the isoform-specific effect of RSKs in normal hematopoiesis using liquid culture assays. We collected transduced cells at day 8, 11, and 14 after transduction and analyzed cell populations by flow cytometry. Similar to colony formation assay results, RSK3 knockdown increased the CD71+ erythroid population by 1.5-fold (p<0.001), but suppressed production of the CD14+ monocyte population. RSK1 knockdown enhanced production of the CD11b+/CD14+ myeloid population, but inhibited the CD71+ erythroid production by 1.5-fold (p<0.001) compared with control HSPCs. There was no significant change in blood cell differentiation in RSK2 knockdown cells. These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs. Disclosures No relevant conflicts of interest to declare.

ScAPAtrap: identification and quantification of alternative polyadenylation sites from single-cell RNA-seq data.

Alternative polyadenylation (APA) generates diverse mRNA isoforms, which contributes to transcriptome diversity and gene expression regulation by affecting mRNA stability, translation and localization in cells. The rapid development of 3' tag-based single-cell RNA-sequencing (scRNA-seq) technologies, such as CEL-seq and 10x Genomics, has led to the emergence of computational methods for identifying APA sites and profiling APA dynamics at single-cell resolution. However, existing methods fail to detect the precise location of poly(A) sites or sites with low read coverage. Moreover, they rely on priori genome annotation and can only detect poly(A) sites located within or near annotated genes. Here we proposed a tool called scAPAtrap for detecting poly(A) sites at the whole genome level in individual cells from 3' tag-based scRNA-seq data. scAPAtrap incorporates peak identification and poly(A) read anchoring, enabling the identification of the precise location of poly(A) sites, even for sites with low read coverage. Moreover, scAPAtrap can identify poly(A) sites without using priori genome annotation, which helps locate novel poly(A) sites in previously overlooked regions and improve genome annotation. We compared scAPAtrap with two latest methods, scAPA and Sierra, using scRNA-seq data from different experimental technologies and species. Results show that scAPAtrap identified poly(A) sites with higher accuracy and sensitivity than competing methods and could be used to explore APA dynamics among cell types or the heterogeneous APA isoform expression in individual cells. scAPAtrap is available at https://github.com/BMILAB/scAPAtrap.

The genomic structure of the highly-conserved dmrt1 gene in Solea senegalensis (Kaup, 1868) shows an unexpected intragenic duplication.

Knowing the factors responsible for sex determination in a species has significant theoretical and practical implications; the dmrt1 gene (Doublesex and Mab-3 (DM)-related Transcription factor 1) plays this role in diverse animal species. Solea senegalensis is a commercially important flat fish in which females grow 30% faster than males. It has 2n = 42 chromosomes and an XX / XY chromosome system for sex determination, without heteromorph chromosomes but with sex proto-chromosome. In the present study, we are providing the genomic structure and nucleotide sequence of dmrt1 gene obtained from cDNA from male and female adult gonads. A cDNA of 2027 containing an open-reading frame (ORF) of 1206 bp and encoding a 402 aa protein it is described for dmrt1 gene of S. senegalensis. Multiple mRNA isoforms indicating a high variable system of alternative splicing in the expression of dmrt1 of the sole in gonads were studied. None isoforms could be related to sex of individuals. The genomic structure of the dmrt1 of S. senegalensis showed a gene of 31400 bp composed of 7 exons and 6 introns. It contains an unexpected duplication of more than 10399 bp, involving part of the exon I, exons II and III and a SINE element found in the sequence that it is proposed as responsible for the duplication. A mature miRNA of 21 bp in length was localized at 336 bp from exon V. Protein-protein interacting networks of the dmrt1 gene showed matches with dmrt1 protein from Cynoglossus semilaevis and a protein interaction network with 11 nodes (dmrt1 plus 10 other proteins). The phylogenetic relationship of the dmrt1 gene in S. senegalensis is consistent with the evolutionary position of its species. The molecular characterization of this gene will enhance its functional analysis and the understanding of sex differentiation in Solea senegalensis and other flatfish.

Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked β-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages

Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.

Temperature-Dependent Alternative Splicing of Precursor mRNAs and Its Biological Significance: A Review Focused on Post-Transcriptional Regulation of a Cold Shock Protein Gene in Hibernating Mammals.

Multiple mRNA isoforms are often generated during processing such as alternative splicing of precursor mRNAs (pre-mRNA), resulting in a diversity of generated proteins. Alternative splicing is an essential mechanism for the functional complexity of eukaryotes. Temperature, which is involved in all life activities at various levels, is one of regulatory factors for controlling patterns of alternative splicing. Temperature-dependent alternative splicing is associated with various phenotypes such as flowering and circadian clock in plants and sex determination in poikilothermic animals. In some specific situations, temperature-dependent alternative splicing can be evoked even in homothermal animals. For example, the splicing pattern of mRNA for a cold shock protein, cold-inducible RNA-binding protein (CIRP or CIRBP), is changed in response to a marked drop in body temperature during hibernation of hamsters. In this review, we describe the current knowledge about mechanisms and functions of temperature-dependent alternative splicing in plants and animals. Then we discuss the physiological significance of hypothermia-induced alternative splicing of a cold shock protein gene in hibernating and non-hibernating animals.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate

BackgroundSome dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis.ResultsWe assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism.ConclusionsOur genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

The role of MAP-kinase p38 in the m. soleus slow myosin mRNA transcription regulation during short-term functional unloading

The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slow-type MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slow-tonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1 myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05). Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus muscles.

Top-ranked expressed gene transcripts of human protein-coding genes investigated with GTEx dataset

With considerable accumulation of RNA-Seq transcriptome data, we have extended our understanding about protein-coding gene transcript compositions. However, alternatively compounded patterns of human protein-coding gene transcripts would complicate gene expression data processing and interpretation. It is essential to exhaustively interrogate complex mRNA isoforms of protein-coding genes with an unified data resource. In order to investigate representative mRNA transcript isoforms to be utilized as transcriptome analysis references, we utilized GTEx data to establish a top-ranked transcript isoform expression data resource for human protein-coding genes. Distinctive tissue specific expression profiles and modulations could be observed for individual top-ranked transcripts of protein-coding genes. Protein-coding transcripts or genes do occupy much higher expression fraction in transcriptome data. In addition, top-ranked transcripts are the dominantly expressed ones in various normal tissues. Intriguingly, some of the top-ranked transcripts are noncoding splicing isoforms, which imply diverse gene regulation mechanisms. Comprehensive investigation on the tissue expression patterns of top-ranked transcript isoforms is crucial. Thus, we established a web tool to examine top-ranked transcript isoforms in various human normal tissue types, which provides concise transcript information and easy-to-use graphical user interfaces. Investigation of top-ranked transcript isoforms would contribute understanding on the functional significance of distinctive alternatively spliced transcript isoforms.

Contrasting activities of estrogen receptor beta isoforms in triple negative breast cancer.

Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERβ) as a possible endocrine therapy target in TNBC. The expression and prognostic effect of ERβ isoforms were analyzed using TCGA breast tumor data, and the expression of ERβ isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERβ2 and ERβ5 were knocked down with siRNA, and ERβ2, ERβ5, and ERβ1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated. ERβ2 and ERβ5 were the predominant endogenous forms of ERβ in TNBC tumors and cell lines. High ERβ2 predicted worse clinical outcome. Knockdown of endogenous ERβ2/ERβ5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERβ2/ERβ5 upregulation did the reverse, increasing survivin and these cell activities. ERβ1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERβ ligands. ERβ2/ERβ5 and ERβ1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERβ isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.

DMD – BIOMARKERS & OUTCOME MEASURES: P.124 mRNA in situ hybridization shows dystrophin transcript subcellular localization imbalance in DMD cells

Dystrophin gene (DMD) internal promoters with their unique first exons generate different mRNA isoforms, of which transcriptional regulation and topography is still poorly defined. We investigated the sub-cellular compartmentalization of DMD transcripts in 17 DMD patients and 2 controls. Five DMD and 1 control cells were immortalized myogenic cells, while 12 DMD and 1 control cells were primary myoblasts. We used two RNAscopeⓇ based probes, which recognize full-length Dp427 transcripts (exons 37-42) or potentially all DMD transcripts (exons 63-75) and DMD transcript localization and quantification was done by RNA/RNA dot counts and by FluiDMD card.

Role of Alternatively Spliced Messenger RNA (mRNA) Isoforms of the Insulin-Like Growth Factor 1 (IGF1) in Selected Human Tumors.

Insulin-like growth factor 1 (IGF1) is a key regulator of tissue growth and development that is also implicated in the initiation and progression of various cancers. The human IGF1 gene contains six exons and five long introns, the transcription of which is controlled by two promoters (P1 and P2). Alternate promoter usage, as well as alternative splicing (AS) of IGF1, results in the expression of six various variants (isoforms) of mRNA, i.e., IA, IB, IC, IIA, IIB, and IIC. A mature 70-kDa IGF1 protein is coded only by exons 3 and 4, while exons 5 and 6 are alternatively spliced code for the three C-terminal E peptides: Ea (exon 6), Eb (exon 5), and Ec (fragments of exons 5 and 6). The most abundant of those transcripts is IGF1Ea, followed by IGF1Eb and IGF1Ec (also known as mechano-growth factor, MGF). The presence of different IGF1 transcripts suggests tissue-specific auto- and/or paracrine action, as well as separate regulation of both of these gene promoters. In physiology, the role of different IGF1 mRNA isoforms and pro-peptides is best recognized in skeletal muscle tissue. Their functions include the development and regeneration of muscles, as well as maintenance of proper muscle mass. In turn, in nervous tissue, a neuroprotective function of short peptides, produced as a result of IGF1 expression and characterized by significant blood-brain barrier penetrance, has been described and could be a potential therapeutic target. When it comes to the regulation of carcinogenesis, the potential biological role of different var iants of IGF1 mRNAs and pro-peptides is also intensively studied. This review highlights the role of IGF1 isoform expression (mRNAs, proteins) in physiology and different types of human tumors (e.g., breast cancer, cervical cancer, colorectal cancer, osteosarcoma, prostate and thyroid cancers), as well as mechanisms of IGF1 spliced variants involvement in tumor biology.