scholarly journals CYCLeR– a novel tool for the full isoform assembly and quantification of circRNAs

2021 ◽  
Author(s):  
Stefan R. Stefanov ◽  
Irmtraud M. Meyer
Keyword(s):  
Splice Variants ◽  
Splice Junction ◽  
Eukaryotic Cell ◽  
The State ◽  
Computational Method ◽  
Full Sequence ◽  
Sequence Identity ◽  
Alternatively Spliced ◽  
Splicing Isoforms ◽  
Biological Patterns

AbstractSplicing is one key mechanism determining the state of any eukaryotic cell. Apart from linear splice variants, circular splice variants (circRNAs) can arise via non-canonical splicing involving a back-splice junction (BSJ). Most existing methods only identify circRNAs via the corresponding BSJ, but do not aim to estimate their full sequence identity or to identify different, alternatively spliced circular isoforms arising from the same BSJ. We here present CYCLeR, the first computational method for identifying the full sequence identify of new and alternatively spliced circRNAs and their abundances while simultaneously co-estimating the abundances of known linear splicing isoforms. We show that CYCLeR significantly out-performs existing methods in terms of sensitivity, precision and quantification of transcripts. When analysing D. melanogaster data, CYCLeR uncovers biological patterns of circRNA expression that other methods fail to observe.

scholarly journals BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Paulo Rapazote-Flores ◽  
Micha Bayer ◽  
Linda Milne ◽  
Claus-Dieter Mayer ◽  
John Fuller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Splice Junction ◽  
Rna Seq ◽  
Rt Pcr ◽  
High Quality ◽  
Transcript Quantification ◽  
Reference Transcript ◽  
Alternatively Spliced ◽  
Time Required ◽  
Comprehensive Reference

Abstract Background The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. Results A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts – BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427–433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20–28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5′ and 3′ UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. Conclusion A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat

1997 ◽  
Vol 272 (4) ◽  
pp. C1250-C1261 ◽  
Author(s):  
B. D. Quednau ◽  
D. A. Nicoll ◽  
K. D. Philipson
Keyword(s):  
Skeletal Muscle ◽  
Polymerase Chain Reaction ◽  
Alternative Splicing ◽  
Reverse Transcriptase ◽  
Splice Variants ◽  
Variable Region ◽  
Intracellular Loop ◽  
Chain Reaction ◽  
Splicing Isoforms ◽  
Polymerase Chain

The gene coding for the Na+/Ca2+ exchanger NCX1 is characterized by a cluster of six exons (A, B, C, D, E, and F) coding for a variable region in the COOH terminus of the large intracellular loop of the protein. Alternative splicing of these exons generates multiple tissue-specific variants of NCX1. Using reverse transcriptase-polymerase chain reaction, we analyzed eight previously described and four new splicing isoforms of NCX1 in a wide variety of tissues and cells. Exons A and B are mutually exclusive, as shown in earlier studies, and splicing isoforms containing exon A are preferentially expressed in heart, brain, and skeletal muscle, whereas splicing variants with exon B are found in all rat tissues except heart. The second and third isoforms of the Na+/Ca2+ exchanger, NCX2 and NCX3, show a deletion of 37 amino acids in the intracellular loop corresponding to parts of the variable region of NCX1. We identified three splicing isoforms of NCX3 in brain and skeletal muscle by reverse transcriptase-polymerase chain reaction. These splice variants are generated by including either of two alternative exons equivalent to the NCX1 exon A or B and by including or excluding a sequence equivalent to the NCX1 exon C. We did not detect any alternative splicing of NCX2. We examined selected tissues from neonatal and adult rats and found developmental regulation for NCX1 and NCX3 splicing isoforms in skeletal muscle. Specific isoform patterns were also detected for NCX1 and NCX3 in cultured cortical neurons, astrocytes, and oligodendrocytes. We suggest a new terminology to distinguish the different splice variants of individual NCX isoforms.

In silicon emergence of an autonomous artificial metabolic system

2019 ◽  
Author(s):  
Shijian Chen
Keyword(s):  
Energy Consumption ◽  
The State ◽  
Computational Method ◽  
Metabolic Function ◽  
Metabolic System ◽  
Biological Structures ◽  
Metabolic Mechanism ◽  
The Right ◽  
Autonomous State ◽  
Shed Light

AbstractIn this work, we establish and evolve an artificial metabolic system in silicon to shed light on how the metabolic mechanism emerged. This system is composed of two subsystems: the artificial genome subsystem (AGS) and the artificial metabolite subsystem (AMS). The whole system is designed to be capable of being autonomous: the dynamics of AGS is capable of situating itself to the dynamics of AMS to provide it with enzymes in the right time and quantity; the dynamics of AMS is capable of implementing the metabolic function and harvest energy so as to pay back the energy consumption of AGS. This kind of autonomous state requires an intricate structure of the AGS. So it is almost impossible to be predetermined manually. With the help of an evolutionary computational method that has a hierarchical mutational structure, the artificial metabolic system with this kind of autonomous state eventually emerged in silicon. We find that ATP and ADP molecules have an important role in making the state of the system autonomous. We also find that the emerged structure of AGS ensemble existing biological structures in the natural cells.

scholarly journals Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

2009 ◽  
Vol 57 (8) ◽  
pp. 763-774 ◽  
Author(s):  
Christine P. Diggle ◽  
Michael Shires ◽  
Derek Leitch ◽  
David Brooke ◽  
Ian M. Carr ◽  
...  
Keyword(s):  
Western Blotting ◽  
Cellular Localization ◽  
Splice Variants ◽  
Immunohistochemical Localization ◽  
Nuclear Staining ◽  
Experimental Artifact ◽  
Alternatively Spliced ◽  
Metabolizing Enzyme ◽  
Knockout Animals

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.

scholarly journals The transformer-2 and fruitless characterisation with developmental expression profiles of sex-determining genes in Bactrocera dorsalis and B. correcta

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kamoltip Laohakieat ◽  
Siriwan Isasawin ◽  
Sujinda Thanaphum
Keyword(s):  
Splice Variants ◽  
Expression Profiles ◽  
Bactrocera Dorsalis ◽  
Developmental Expression ◽  
Alternative Promoters ◽  
Downstream Effector ◽  
Effector Genes ◽  
Early Embryos ◽  
Alternatively Spliced ◽  
Tephritid Fruit Flies

Abstract Sex determination in tephritid fruit flies involves a signaling cascade of alternatively spliced genes. The Transformer (TRA) and Transformer-2 (TRA-2) complex establishes an autoregulatory loop switching sex-specific splicing of tra pre-mRNA in females. The TRA/TRA-2 complex also regulates the sex-specific splicing of downstream effector genes, doublesex (dsx) and fruitless (fru). In Ceratitis capitata, a Maleness-on the-Y (MoY) gene modulates sex-specifically spliced Cctra pre-mRNA and results in the breakdown of the Cctra autoregulatory loop in males. In this study, the tra-2 and fru genes were characterised in two key pests, Bactrocera dorsalis and B. correcta. The tra-2 genes showed high degrees of conservation among tephritids. The complex gene organisation for each of Bdfru and Bcfru were identified. There are sex-specific and non sex-specific transcripts generated by alternative promoters as found in Drosophila melanogaster and other insects. RNAi knockdown of Bdtra transcripts showed that BdTRA controls the sex-specific splicing of Bddsx and Bdfru pre-mRNAs. Developmental expression analysis shows that multiple splice variants of Bdtra and Bctra RNAs are present before and during cellular blastoderm formation and that the mature sex-specific variants become fixed later in embryogenesis. Furthermore, the BddsxM splice variants are found in early embryos at the beginning of gastulation, but BdfruM does not appear until the larval stage. We proposed that the zygotic tra loop is initiated in both female and male embryos before becoming automatised or abolished by MoY, respectively.

scholarly journals Allosteric modulation of alternatively spliced Ca2+-activated Cl−channels TMEM16A by PI(4,5)P2and CaMKII

2020 ◽  
Vol 117 (48) ◽  
pp. 30787-30798
Author(s):  
Woori Ko ◽  
Seung-Ryoung Jung ◽  
Kwon-Woo Kim ◽  
Jun-Hee Yeon ◽  
Cheon-Gyu Park ◽  
...  
Keyword(s):  
Binding Site ◽  
In Silico ◽  
Single Channel ◽  
Splice Variants ◽  
Noise Analysis ◽  
Mechanistic Explanation ◽  
Intracellular Loop ◽  
Open Probability ◽  
In Silico Simulation ◽  
Alternatively Spliced

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl−channel activated by intracellular Ca2+and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P2sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

scholarly journals Identification of embryo specific human isoforms using a database of predicted alternative splice forms

2006 ◽  
Vol 3 (1) ◽  
pp. 1-10
Author(s):  
Heike Pospisil
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Splice Variants ◽  
Main Idea ◽  
Protein Isoforms ◽  
Unequal Distribution ◽  
Interesting Candidate ◽  
Alternatively Spliced ◽  
Specific Alternative ◽  
Splice Forms

Abstract Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from a small number of genes. Its study became one of the hot topics in computational genome analysis. The repository EASED (Extended Alternatively Spliced EST Database, http://eased.bioinf.mdc-berlin.de/) stores a large collection of splice variants predicted from comparing the human genome against EST databases. It enables finding new unpublished splice forms that could be interesting candidate genes for stage specific, diseases specific or tissue specific splicing. The main idea behind selecting specific splice forms is to compare the number and the origin of ESTs confirming one isoform with the number and the origin of ESTs confirming the opposite isoform. A measure asDcs is introduced to take into account the unequal distribution of ESTs per splice site on one hand, and the possible uncertainties due to the relatively low quality of EST data on the other hand. First, the number of ESTs per splice site is scaled with a modified Hill function. The measure asDcs computes in the second step the distance of each pair of ESTs from equipartition. Equipartition exists if for every number of adult ESTs the same number of embryonic ESTs. The effect of several input parameters for the scaling of true EST values is analysed and can be reproduced on http://cardigan.zbh.uni-hamburg.de/asDcs. Some of the obtained best scoring hits for selected parameters (transcription factor P65, drebrin, and fetuin) have been already described in literature as been involved in embryonic development. This result shows the plausibility of this approach and looks promising for the identification of unplublished embryo specific alternative splice sites in human.

scholarly journals Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3

1997 ◽  
Vol 328 (2) ◽  
pp. 549-558 ◽  
Author(s):  
Elaine HUSTON ◽  
Simon LUMB ◽  
Annette RUSSELL ◽  
Cath CATTERALL ◽  
H. Annette ROSS ◽  
...  
Keyword(s):  
High Speed ◽  
Splice Variants ◽  
Short Form ◽  
Amino Acid Identity ◽  
Open Reading Frame ◽  
Cytosol Fraction ◽  
Reading Frame ◽  
Alternatively Spliced ◽  
Camp Hydrolysis ◽  
Rapid Amplification

5ʹ-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5ʹ sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue ‘long-form’ PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 μM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 μM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 μM) to rolipram inhibition than its cytosolic form (IC50 0.2 μM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.

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