scholarly journals A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene

2003 ◽  
Vol 374 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Daesung SHIN ◽  
Sangjin PARK ◽  
Chankyu PARK
Keyword(s):  
Alternative Splicing ◽  
Glutamine Synthetase ◽  
Genomic Structure ◽  
Cell Types ◽  
Alternative Transcript ◽  
Exon Trapping ◽  
Exon 1 ◽  
Rapid Amplification ◽  
Altered Form ◽  
Human And Mouse

The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5′-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase–PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1′. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5′-untranslated region (UTR) containing the exon 1′. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin–Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5′-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5′-UTRs, demonstrating that the long form with extra 5′-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5′-UTRs were less efficient, showing 6–8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1′, the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5′-UTR is, to some extent, due to an abortive translation from the upstream ATG.

scholarly journals Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Luyi Tian ◽  
Jafar S. Jabbari ◽  
Rachel Thijssen ◽  
Quentin Gouil ◽  
Shanika L. Amarasinghe ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Single Cells ◽  
Transcript Level ◽  
Cell Types ◽  
Alternative Transcript ◽  
Long Read ◽  
Different Cell Types ◽  
Human And Mouse ◽  
Comprehensive Characterization

AbstractA modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

Cloning and Expression of a Short Fas Ligand: A New Alternatively Spliced Product of the Mouse Fas Ligand Gene

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3456-3467 ◽  
Author(s):  
Emira Ayroldi ◽  
Francesca D’Adamio ◽  
Ornella Zollo ◽  
Massimiliano Agostini ◽  
Rosalba Moraca ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
Fas Ligand ◽  
Cell Types ◽  
Cloning And Expression ◽  
Activated T Cells ◽  
Rnase Protection ◽  
Reading Frame ◽  
Tumor Necrosis Factor Ligand ◽  
Exon 1

The Fas/FasL system mediates apoptosis in several different cell types, including T lymphocytes. Fas ligand (FasL), a 40-kD type II membrane protein also expressed in activated T cells, belongs to the tumor necrosis factor ligand family. We describe a new alternative splicing of mouse FasL, named FasL short (FasLs), cloned by reverse transcriptase-polymerase chain reaction. FasLs is encoded by part of exon 1 and part of exon 4 of FasL gene. The protein encoded by FasLs mRNA has a putative initiation code at position 756 and preserves the same reading frame as FasL, resulting in a short molecule lacking the intracellular, the transmembrane, and part of the extracellular domains. RNase protection and immunoprecipitation analysis showed that FasLs is expressed in nonactivated normal spleen cells and in hybridoma T cells and that it is upregulated upon activation by anti-CD3 monoclonal antibody (MoAb). Moreover, FasLs-transfected cells expressed soluble FasLs in the supernatant and became resistant to apoptosis induced by agonist anti-Fas MoAb. Thus, FasLs, a new alternative splicing of FasL, is involved in the regulation of Fas/FasL-mediated cell death.

scholarly journals Coverage-dependent bias creates the appearance of binary splicing in single cells

2019 ◽  
Author(s):  
Carlos F. Buen Abad Najar ◽  
Nir Yosef ◽  
Liana F. Lareau
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Rna Splicing ◽  
Single Cells ◽  
Cell Types ◽  
Rna Seq ◽  
Unique Identity ◽  
Underlying Reality ◽  
Human And Mouse ◽  
Insight Into

Single cell RNA sequencing provides powerful insight into the factors that determine each cell’s unique identity, including variation in transcription and RNA splicing among diverse cell types. Previous studies led to the surprising observation that alternative splicing outcomes among single cells are highly variable and follow a bimodal pattern: a given cell consistently produces either one or the other isoform for a particular splicing choice, with few cells producing both isoforms. Here we show that this pattern arises almost entirely from technical limitations. We analyzed single cell alternative splicing in human and mouse single cell RNA-seq datasets, and modeled them with a probablistic simulator. Our simulations show that low gene expression and low capture efficiency distort the observed distribution of isoforms in single cells. This gives the appearance of a binary isoform distribution, even when the underlying reality is consistent with more than one isoform per cell. We show that accounting for the true amount of information recovered can produce biologically meaningful measurements of splicing in single cells.

Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes

1992 ◽  
Vol 67 (02) ◽  
pp. 272-276 ◽  
Author(s):  
C Paul ◽  
E van der Logt ◽  
Pieter H Reitsma ◽  
Rogier M Bertina
Keyword(s):  
Alternative Splicing ◽  
Tissue Factor ◽  
Stop Codon ◽  
Cell Types ◽  
Northern Analysis ◽  
Human Monocytes ◽  
Mrna Species ◽  
Protein Levels ◽  
Intron 1 ◽  
Tf Gene

SummaryAlthough normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Molecule ◽  
High Fat Diet ◽  
Human Heart ◽  
Full Length ◽  
Funding Source ◽  
Smrt Sequencing ◽  
High Fat ◽  
Exon 1 ◽  
Novel Exon

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Exon-1 skipping and intron-1 retaining by alternative splicing of the c-KIT gene encodes a novel splice variant in the skin of Merino sheep (Ovis aries)

2021 ◽  
Author(s):  
Siva Arumugam Saravanaperumal ◽  
Stefano Pallotti ◽  
Dario Pediconi ◽  
Carlo Renieri ◽  
Antonietta La Terza
Keyword(s):  
Alternative Splicing ◽  
Splice Variant ◽  
Ovis Aries ◽  
Merino Sheep ◽  
Intron 1 ◽  
Kit Gene ◽  
Exon 1

Genomic structure, chromosome location, and alternative splicing of the humanNKG2A gene

1996 ◽  
Vol 44 (4) ◽  
pp. 286-291 ◽  
Author(s):  
Béatrice Plougastel ◽  
Tania Jones ◽  
John Trowsdale
Keyword(s):  
Alternative Splicing ◽  
Genomic Structure ◽  
Chromosome Location

scholarly journals Comparing the electrophysiology and morphology of human and mouse layer 2/3 pyramidal neurons with Bayesian networks

2020 ◽  
Author(s):  
Bojan Mihaljević ◽  
Pedro Larrañaga ◽  
Concha Bielza
Keyword(s):  
Bayesian Networks ◽  
Probabilistic Reasoning ◽  
Pyramidal Neurons ◽  
Temporal Cortex ◽  
Input Resistance ◽  
Cell Types ◽  
Dendritic Arbor ◽  
Basal Dendrites ◽  
Morphological Variables ◽  
Human And Mouse

ABSTRACTPyramidal neurons are the most common neurons in the cerebral cortex. Understanding how they differ between species is a key challenge in neuroscience. We compared human temporal cortex and mouse visual cortex pyramidal neurons from the Allen Cell Types Database in terms of their electrophysiology and basal dendrites’ morphology. We found that, among other differences, human pyramidal neurons had a higher threshold voltage, a lower input resistance, and a larger basal dendritic arbor. We learned Gaussian Bayesian networks from the data in order to identify correlations and conditional independencies between the variables and compare them between the species. We found strong correlations between electrophysiological and morphological variables in both species. One result is that, in human cells, dendritic arbor width had the strongest effect on input resistance after accounting for the remaining variables. Electrophysiological variables were correlated, in both species, even with morphological variables that are not directly related to dendritic arbor size or diameter, such as mean bifurcation angle and mean branch tortuosity. Contrary to previous results, cortical depth was correlated with both electrophysiological and morphological variables, and its effect on electrophysiological could not be explained in terms of the morphological variables. Overall, the correlations among the variables differed strikingly between human and mouse neurons. Besides identifying correlations and conditional independencies, the learned Bayesian networks might be useful for probabilistic reasoning regarding the morphology and electrophysiology of pyramidal neurons.

scholarly journals Alternative Splicing of Transcription Factors' Genes: Beyond the Increase of Proteome Diversity

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
David Talavera ◽  
Modesto Orozco ◽  
Xavier de la Cruz
Keyword(s):  
Transcription Factors ◽  
Alternative Splicing ◽  
Expression Patterns ◽  
Developmental Changes ◽  
Functional Families ◽  
Transcription Regulators ◽  
Alternatively Spliced ◽  
The Impact ◽  
Human And Mouse

Functional modification of transcription regulators may lead to developmental changes and phenotypical differences between species. In this work, we study the influence of alternative splicing on transcription factors in human and mouse. Our results show that the impact of alternative splicing on transcription factors is similar in both species, meaning that the ways to increase variability should also be similar. However, when looking at the expression patterns of transcription factors, we observe that they tend to diverge regardless of the role of alternative splicing. Finally, we hypothesise that transcription regulation of alternatively spliced transcription factors could play an important role in the phenotypical differences between species, without discarding other phenomena or functional families.

scholarly journals The sperm nucleus: chromatin, RNA, and the nuclear matrix

Reproduction ◽  
2011 ◽  
Vol 141 (1) ◽  
pp. 21-36 ◽  
Author(s):  
Graham D Johnson ◽  
Claudia Lalancette ◽  
Amelia K Linnemann ◽  
Frédéric Leduc ◽  
Guylain Boissonneault ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Genomic Structure ◽  
Sperm Nucleus ◽  
Nuclear Volume ◽  
Clinical Markers ◽  
Paternal Genome ◽  
Epigenetic Effects ◽  
History Of ◽  
Low Levels ◽  
Human And Mouse

Within the sperm nucleus, the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm, they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such, they may soon be utilized as clinical markers of male fertility. In this review, we explore and discuss how this may be orchestrated.

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