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At least 19 recordsLinked to original sources
PubMed2025

Decipher RNA isoform combinations from minigene splicing assays and massive parallel sequencing with MAGIC.

SUMMARY: Functional testing of RNA using minigene splicing assays is increasingly being realized to demonstrate the effects of variants on splicing. In complex cases, variant pathogenicity is assessed by Sanger sequencing, which can be time consuming and may be replaced by short read sequencing. Moreover, strategies based on long read sequencing of the amplified minigene construct are promising and allow the isoforms to be fully characterized. We introduce MAGIC, a user-friendly tool that first generates the artificial construction genome files required to then perform alignment, assembly and annotation of the isoforms obtained by either short or long read minigene splicing assay sequencing. AVAILABILITY AND IMPLEMENTATION: MAGIC is available at https://github.com/LBGC-CFB/MAGIC. Zenodo DOI: 10.5281/zenodo.17052752.

High-Throughput Nucleotide Sequencing↗
PubMed1992

Multiple Rh messenger RNA isoforms are produced by alternative splicing.

Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.

Amino Acid Sequence↗
PubMed2025

Perplexity as a Metric for Isoform Diversity in the Human Transcriptome.

Long-read sequencing (LRS) has revealed a far greater diversity of RNA isoforms than earlier technologies, increasing the critical need to determine which, and how many, isoforms per gene are biologically meaningful. To define the space of relevant isoforms from LRS, many existing analysis pipelines rely on arbitrary expression cutoffs, but a single threshold cannot accommodate the broad variability in isoform complexity across genes, cell-types, and disease states captured by LRS. To address this, we propose using perplexity-an interpretable measure derived from entropy-that quantifies the effective number of isoforms per gene based on the full, unfiltered isoform ratio distribution. Calculating perplexity for 124 ENCODE4 PacBio LRS datasets spanning 55 human cell types, we show that it provides intuitive assessments of isoform diversity and captures uncertainty across genes with varying complexity. Perplexity can be calculated at multiple gene regulatory levels-from transcript to protein-to compare how isoform diversity is reduced across stages of gene expression. On average, genes have an ORF-level perplexity of 2.1, indicating production of two distinct protein isoforms. We extended this analysis to evaluate expression variation across tissues and identified 4,593 ORFs across 3,102 genes with moderate to extreme tissue-specificity. We propose perplexity as a consistent, quantitative metric for interpreting isoform diversity across genes, cell types, and disease states. All results are compiled into a community resource to enable cross-study comparisons of novel isoforms.

Journal Article↗
PubMed1988

Isoform-specific modulation of Na+, K+-ATPase alpha-subunit gene expression in hypertension.

Sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and myocardial contractility. By means of RNA blot analyses of cardiac, aortic, and skeletal muscle RNAs in two rat hypertensive models, Na+,K+-ATPase alpha-subunit messenger RNA isoforms (alpha 2 and alpha 3) were shown to be deinduced in response to increased intravascular pressure. The changes were observed after 48 hours or more of experimental hypertension. Under these conditions, there is coordinate induction of another alpha isoform (alpha 1) and of beta-subunit messenger RNAs, probably in response to alterations in sodium flux rather than to elevated blood pressure.

Angiotensin II↗
PubMedDate not supplied

Comparison of alpha-tropomyosin sequences from smooth and striated muscle.

Tropomyosins are a closely related family of proteins with a dimeric alpha-coiled-coil structure. Skeletal isoforms are composed of two types of subunits, alpha and beta which, in turn, are assorted into two main molecular species alpha alpha and alpha beta. Both isoforms are present in different molar ratios in individual skeletal muscle types. In small mammals, however, only alpha-chain is expressed in cardiac muscle. Tropomyosin, in association with the troponin complex (troponin-I, -T and -C) plays a central role in the Ca2+-dependent regulation of vertebrate striated muscle contraction. On the other hand, despite structural similarities with the striated isoforms, the function of this protein in smooth muscle and non-muscle cells remains unknown, because in these cells contraction is thought to be regulated by myosin-linked processes independently of tropomyosin. Here we report the nucleotide sequences of cloned complementary DNAs for rat striated and smooth muscle alpha-tropomyosin. Comparison of the derived amino-acid sequences reveals the existence of tissue-specific peptides that delimit the putative troponin-I and troponin-T binding domains of tropomyosin. S1-nuclease mapping studies reveal the existence of three distinct alpha-tropomyosin messenger RNA isoforms each encoding a different protein; these isoforms are tissue-specific, developmentally regulated and most probably encoded by the same gene.

Amino Acid Sequence↗
PubMed1989

Interferon gamma inhibits both proliferation and expression of differentiation-specific alpha-smooth muscle actin in arterial smooth muscle cells.

Differentiation of muscle cells is characterized morphologically by the acquisition of contractile filaments and characteristic shape changes, and on the molecular level by induction of the expression of several genes, including those for the muscle-specific alpha-actin isoforms. IFN-gamma is an inhibitor of proliferation for several cells, including vascular smooth muscle, and is also an inducer of differentiated properties for several hematopoietic cells. We have therefore investigated whether IFN-gamma affects the expression of alpha-smooth muscle actin in cultured arterial smooth muscle cells. Cells exposed to IFN-gamma show a reduction of alpha-smooth muscle actin-containing stress fibers, as detected by immunofluorescence. The effect was observed in all phases of the cell cycle, and was caused by a reduction of the synthesis of alpha-smooth muscle actin protein as revealed by two-dimensional electrophoretic analysis of actin isoforms. RNA hybridization using a cRNA probe that hybridizes to all actin mRNAs showed that IFN-gamma-treated cells have a reduced content of the 1.7-kb mRNA that codes for alpha-smooth muscle actin, and to a lesser extent, also of the 2.1-kb mRNA encoding the beta and gamma-cytoplasmic actins. The reduction of alpha-smooth muscle actin mRNA was confirmed using an alpha-smooth muscle actin-specific cRNA probe. The reduction of alpha-smooth muscle actin mRNA occurs within 12 h, and is dependent on protein synthesis, since cycloheximide treatment reversed the effect. The inhibition of this mRNA species was dose dependent, and detectable by RNA hybridization at a dose of 50 U/ml IFN-gamma. These results suggest that the differentiation of arterial smooth muscle cells is not necessarily coupled to an inhibition of cellular proliferation. Instead, IFN-gamma may regulate the expression of several genes that control both proliferation and expression of differentiation markers.

Actin Cytoskeleton↗
PubMed1992

Avian cardiac tropomyosin gene produces tissue-specific isoforms through alternative RNA splicing.

We have isolated a quail cardiac tropomyosin gene which encodes three distinct isoforms through the use of alternative exon splicing. Characterization of cDNA clones produced by this gene indicate that the gene encodes a unique 284 amino acid cardiac tropomyosin isoform, along with a 248 amino acid cytoskeletal and 284 amino acid smooth muscle isoforms. Northern analyses indicate that the gene is primarily expressed in cardiac muscle, with only minor expression of the cytoskeletal and smooth muscle transcripts.

Amino Acid Sequence↗
PubMed1991

Changes in beta-tubulin isoforms and their RNA level in synchronized tobacco cells.

Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their beta-tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the beta-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot beta-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that beta-tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at M phase and decreased during G1 phase.

Aphidicolin↗
PubMed1987

Myosin heavy chain messenger RNA and protein isoform transitions during cardiac hypertrophy. Interaction between hemodynamic and thyroid hormone-induced signals.

Expression of the cardiac myosin isozymes is regulated during development, by hormonal stimuli and hemodynamic load. In this study, the levels of expression of the two isoforms (alpha and beta) of myosin heavy chain (MHC) during cardiac hypertrophy were investigated at the messenger RNA (mRNA) and protein levels. In normal control and sham-operated rats, the alpha-MHC mRNA predominated in the ventricular myocardium. In response to aortic coarctation, there was a rapid induction of the beta-MHC mRNA followed by the appearance of comparable levels of the beta-MHC protein in parallel to an increase in the left ventricular weight. Administration of thyroxine to coarctated animals caused a rapid deinduction of beta-MHC and induction of alpha-MHC, both at the mRNA and protein levels, despite progression of left ventricular hypertrophy. These results suggest that the MHC isozyme transition during hemodynamic overload is mainly regulated by pretranslational mechanisms, and that a complex interplay exists between hemodynamic and hormonal stimuli in MHC gene expression.

Animals↗
PubMed1985

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals.

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.

Amino Acid Sequence↗
PubMed1991

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Microdiversity of human-plasma-membrane calcium-pump isoform 2 generated by alternative RNA splicing in the N-terminal coding region.

cDNA species covering the entire coding sequence of the human homologue of the rat plasma membrane Ca(2+)-ATPase (PMCA) isoform 2 have been isolated and characterized. The deduced amino acid sequence shows 99% identity with that of the rat protein and can be aligned with the latter without gaps except for one 14-amino-acid-residue insert in the region immediately preceding the putative phospholipid-sensitive domain in the human pump. cDNA clones isolated by anchored polymerase-chain reaction revealed additional microheterogeneity in the same N-terminal PMCA2-coding region. Alternative RNA splicing involving a region of 135 nucleotides generates three types of cDNA. One does not contain any of the 135 bp, and the other two contain 42 bp or the entire 135 bp of the optional sequence. Analysis of genomic DNA indicates that this sequence is encoded by three separate exons of 33, 60 and 42 bp. Although each of these exons could be inserted into the mRNA without changing the reading frame, polymerase-chain amplifications using cDNA libraries from several human tissues show that the 33-bp and the 60-bp exons are never independently used during splicing. The unequal distribution of the splice variants suggests tissue-specific regulation of the alternative-splicing pathways and indicates a functional specialization of the encoded isoform subtypes.

Amino Acid Sequence↗
PubMed2025

Bayesian identification of differentially expressed isoforms using a novel joint model of RNA-seq data.

We develop a Bayesian approach, BayesIso, to identify differentially expressed isoforms from RNA-seq data. The approach features a novel joint model of the sample variability and the deferential state of isoforms. Specifically, the within-sample variability and the between-sample variability of each isoform are modeled by a Poisson-Lognormal model and a Gamma-Gamma model, respectively. Using a Bayesian framework, the differential state of each isoform and the model parameters are jointly estimated by a Markov Chain Monte Carlo (MCMC) method. Extensive studies using simulation and real data demonstrate that BayesIso can effectively detect isoforms of less differentially expressed and differential transcripts for genes with multiple isoforms. We applied the approach to breast cancer RNA-seq data and uncovered a unique set of isoforms that form key pathways associated with breast cancer recurrence. First, PI3K/AKT/mTOR signaling and PTEN signaling pathways are identified as being involved in breast cancer development. Further integrated with protein-protein interaction data, pathways of Jak-STAT, mTOR, MAPK and Wnt signaling are revealed in association with breast cancer recurrence. Finally, several pathways are activated in the early recurrence of breast cancer. In tumors that occur early, members of pathways of cellular metabolism and cell cycle (such as CD36 and TOP2A) are upregulated, while immune response genes such as NFATC1 are downregulated.

Humans↗
PubMed1990

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA.

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern.

Amino Acid Sequence↗
PubMed2026

circASbase: A Comprehensive Database of Alternative Splicing Events in circRNAs.

Although extensive evidence has underscored the critical role of alternative splicing (AS) in generating mature circular RNA (circRNA) isoforms and augmenting their functional diversity, a significant gap remains in the availability of specialized databases housing circRNA AS events. To bridge this gap, we develop circASbase, a pioneering and comprehensive database that catalogs 452,129 AS events in 884,047 full-length circRNAs from 581 samples across 13 species, and provides rich annotations to facilitate understanding the splicing regulation of circRNA. Our findings reveal substantial differences between circRNAs and linear transcripts regarding the distribution and occurrence of AS events, highlighting the unique regulatory landscape of circRNAs. These special splicing events result in functional differences of circRNAs by affecting internal ribosome entry sites, N6-methyladenosine sites, open reading frames, protein features, microRNA targets, and more. In summary, circASbase not only meets the urgent need of the research community for data repositories, but also represents a significant advancement in our understanding of circRNA biology. With its user-friendly interfaces and web-based visualization tools, circASbase is poised to become an indispensable resource for researchers exploring the regulatory mechanisms and functional roles of AS events in circRNAs. This database will continuously drive new insights and discoveries in the field, setting the stage for further advancements in circRNA research. circASbase is freely available at http://reprod.njmu.edu.cn/cgi-bin/circASbase/.

Alternative Splicing↗
PubMed2014

Expression of ZNF804A in human brain and alterations in schizophrenia, bipolar disorder, and major depressive disorder: a novel transcript fetally regulated by the psychosis risk variant rs1344706.

IMPORTANCE: The single-nucleotide polymorphism rs1344706 in the zinc finger protein 804A gene (ZNF804A) shows genome-wide association with schizophrenia and bipolar disorder. Little is known regarding the expression of ZNF804A and the functionality of rs1344706. OBJECTIVES: To characterize ZNF804A expression in human brain and to investigate how it changes across the life span and how it is affected by rs1344706, schizophrenia, bipolar disorder, and major depressive disorder. DESIGN, SETTING, AND PARTICIPANTS: Molecular and immunochemical methods were used to study ZNF804A messenger RNA (mRNA) and ZNF804A protein, respectively. ZNF804A transcripts were investigated using next-generation sequencing and polymerase chain reaction-based methods, and ZNF804A protein was investigated using Western blots and immunohistochemistry. Samples of dorsolateral prefrontal cortex and inferior parietal lobe tissue were interrogated from 697 participants between 14 weeks' gestational age and age 85 years, including patients with schizophrenia, bipolar disorder, or major depressive disorder. MAIN OUTCOMES AND MEASURES: Quantitative measurements of ZNF804A mRNA and immunoreactivity, and the effect of diagnosis and rs1344706 genotype. RESULTS: ZNF804A was expressed across the life span, with highest expression prenatally. An abundant and developmentally regulated truncated ZNF804A transcript was identified, missing exons 1 and 2 (ZNF804AE3E4) and predicted to encode a protein lacking the zinc finger domain. rs1344706 influenced expression of ZNF804AE3E4 mRNA in fetal brain (P&#x2009;=&#x2009;.02). In contrast, full-length ZNF804A showed no association with genotype (P&#x2009;>&#x2009;.05). ZNF804AE3E4 mRNA expression was decreased in patients with schizophrenia (P&#x2009;=&#x2009;.006) and increased in those with major depressive disorder (P&#x2009;<&#x2009;.001), and there was a genotype-by-diagnosis interaction in bipolar disorder (P&#x2009;=&#x2009;.002). ZNF804A immunoreactivity was detected in fetal and adult human cerebral cortex. It was localized primarily to pyramidal neurons, with cytoplasmic as well as dendritic and nuclear staining. No differences in ZNF804A-immunoreactive neurons were seen in schizophrenia or related to rs1344706 (P&#x2009;>&#x2009;.05). CONCLUSIONS AND RELEVANCE: rs1344706 influences the expression of ZNF804AE3E4, a novel splice variant. The effect is limited to fetal brain and to this isoform. It may be part of the mechanism by which allelic variation in ZNF804A affects risk of psychosis. ZNF804A is translated in human brain, where its functions may extend beyond its predicted role as a transcription factor.

Adolescent↗
PubMed1990

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos.

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.

Actins↗