RNA-seq Analysis
Altuna Akalin in Computational Genomics with R, 2020
RNA-seq generates valuable data that contains information not only at the gene level but also at the level of exons and transcripts. Moreover, the kind of information that we can extract from RNA-seq is not limited to expression quantification. It is possible to detect alternative splicing events such as novel isoforms (Trapnell et al., 2010), and differential usage of exons (Anders et al., 2012). It is also possible to observe sequence variants (substitutions, insertions, deletions, RNA-editing) that may change the translated protein product (McKenna et al., 2010). In the context of cancer genomes, gene-fusion events can be detected with RNA-seq (McPherson et al., 2011). Finally, for the purposes of gene prediction or improving existing gene predictions, RNA-seq is a valuable method (Stanke and Morgenstern, 2005). In order to learn more about how to implement these, it is recommended that you go through the tutorials of the cited tools.
Molecular and Cellular Pathogenesis of Systemic Lupus Erythematosus
Richard K. Burt, Alberto M. Marmont in Stem Cell Therapy for Autoimmune Disease, 2019
Although the precise molecular mechanisms underlying ζ chain deficiency is still being examined, current evidence supports the possibility of a transcriptional defect. In SLE, T cells that expressed low levels of T cell receptor ζ chain transcripts, cloning and sequencing revealed more frequent heterogeneous polymorphisms/ mutations and alternative splicing of T cell receptor ζ chain.12,23,24 Most of these mutations are localized to the three immunoreceptor tyrosine activation motifs (ITAM) or guanosine triphosphate (GTP) binding domain and could functionally affect the ζ chain providing a molecular basis to the known T cell signaling abnormalities in SLE T cells. Absence of the mutations/ polymorphisms in the genomic DNA suggests that these are the consequence of irregular RNA editing. SLE patients also showed significant increase in the splice variation of the ζ chain. The splicing abnormality included two insertion splice variants of 145 bases and 93 bases between exons I and II, and also several deletion splice variants of T cell recceptor ζ chain resulting from the deletion of individual exons II, VI, VII, or a combined deletion of exons V and VI; VI and VII; II, III and IV; and V, VI and VII in SLE T cells.
Genetics of Wilms tumor
J. K. Cowell in Molecular Genetics of Cancer, 2003
The WT1 gene encodes a developmentally regulated transcription factor (Figure 1). The gene of 10 exons, spanning approximately 50 kb, is transcribed to a 3.5 kb mRNA that undergoes alternative splicing. The WT1 protein (52–54 kD, predicted Mr 46 kD) contains a carboxyterminal DNA-binding domain with four zinc-fingers of the Cys2-His2 type, and an amino-terminal transactivation domain rich in proline, glutamine, serine, and lysin. The N-terminal part of the protein was also shown to be responsible for self-dimerization (Moffett et al., 1995; Reddy et al., 1995). Mutations within the zinc-finger domain found in Wilms tumor and DDS destroy the DNA-binding ability of WT1 (Haber et al., 1990; Pelletier et al., 1991), whereas the properties of the transactivation domain can be changed by mutations to confer activation instead of repression of some target promoters (Park et al., 1993b, 1993c). Two alternative splice sites give rise to four WT1 isoforms. Exon 5 can be variably spliced resulting in the presence or absence of 17 amino acids in the amino-terminal part of the protein but this appears to be specific to mammals. Usage of the second alternative splice site at the end of exon 9, that is also present in lower vertebrates, determines the presence of three amino acids, lysine-threonine-serine (KTS), between zinc-fingers 3 and 4 (Rauscher, 1993). Mutations within the +/−KTS splice junction, which upset the balance of WT1 isoforms, are the underlying cause of severe genito-urinary abnormalities seen in Frasier syndrome (Barbaux et al., 1997). RNA editing that results in a proline to leucine change at position 280, and the usage of an alternative translation start, adding 68 amino acids to WT1, increases the number of potential isoforms to 16 (Bruening and Pelletier, 1996; Sharma et al., 1994). Further complexity may be added to the regulation of WT1 at the post-transcriptional level, since phosphorylation of the WT1 protein isoforms was shown to inhibit DNA-binding and alter WT1 transcriptional activity and cellular translocation (Sakamoto et al., 1997; Ye et al., 1996).
Is subretinal AAV gene replacement still the only viable treatment option for choroideremia?
Published in Expert Opinion on Orphan Drugs, 2021
Ruofan Connie Han, Lewis E. Fry, Ariel Kantor, Michelle E. McClements, Kanmin Xue, Robert E. MacLaren
Finally, CRISPR-directed RNA editing represents another novel approach to targeted correction of single nucleotide variants, in RNA rather than DNA [54]. A wide variety of approaches have been developed to edit RNA in vitro. Each approach currently uses a variant of the Adenosine Deaminase Acting on RNA (ADAR), naturally expressed enzymes in human cells that undertake physiological RNA editing functions. These deaminases convert adenosine bases to inosine in RNA, which is read as a guanosine in cellular processes such as translation and splicing [63]. This effectively creates an A > G edit in RNA and can be harnessed for the correction of G > A mutations. ADAR variants have also been engineered to create C > U edits: together, they can theoretically address up to 10% of known CHM mutations [64,65]. Harnessed for site-directed RNA editing, ADAR can be recruited to editing sites of interest by systems that link ADAR to an effector molecule and direct the ADAR-effector system with a gRNA to the base to be edited [54]. Many effectors have been developed including those based on CRISPR-Cas13 [65,66] or Cas9 systems [67], bacteriophage-derived λN peptide [71] and BoxB system, aptamer-like systems such as the MS2 bacteriophage coat protein (MCP) or GluR2 system [69]. Additionally, systems that deliver only a gRNA and use only endogenously expressed ADAR have been developed [69–71], in contrast [68] to other systems that require ADAR overexpression.
Significance of DopEcR, a G-protein coupled dopamine/ecdysteroid receptor, in physiological and behavioral response to stressors
Published in Journal of Neurogenetics, 2020
Emily Petruccelli, Arianna Lark, James A. Mrkvicka, Toshihiro Kitamoto
The DopEcR gene spans a 12,739 bp region on the left arm of the third chromosome in D. melanogaster. Three different DopEcR transcripts are currently known, which differ in 5’ and 3’ UTR sequences, but each encodes for the same 322 aa protein sequence. Interestingly, at least 25 different RNA editing sites have been identified between DopEcR cDNA and EST sequences (Brody & Cravchik, 2000), but their functional implication remains to be determined. Phylogenetic analysis of the DopEcR protein sequence indicates that it shares greater similarity to vertebrate β-adrenergic receptors than it does to the fly dopaminergic receptor family (Srivastava et al., 2005), suggesting that DopEcR may have unique non-redundant dopaminergic functions compared to the other fly dopamine receptors.
RNA A-to-I editing, environmental exposure, and human diseases
Published in Critical Reviews in Toxicology, 2021
Akin Cayir
RNA editing is a unique type of RNA modification and occurs in the specific nucleic acids of RNAs after transcription. There are two common forms of RNA editing in mammals including Adenosine to Inosine (A-to-I) and Cytidine to Uridine (C-to-U) conversion. RNA editing was initially discovered in the embryos of African clawed frog, Xenopus laevis, more than 30 years ago (Bass BL and Weintraub 1987; Rebagliati and Melton 1987). The first finding in human tissues was reported in 1987, regarding C-to-U conversion in the human mRNA of apolipoprotein-B48 gene in the intestine which was suggested as a tissue-specific modification of a single mRNA nucleotide (Powell et al. 1987). After these preliminary findings, RNA editing has been documented over three decades, leaving some aspects unexplored. Today, like several RNA modifications, such as m6A, RNA editing is associated with various diseases with limited findings. Furthermore, a few studies indicated that environmental stress could affect the RNA edition (Dorn et al. 2019). Together, we review a general overview of RNA editing- environmental exposures associations and RNA editing-diseases associations (cancer). This work presents initial evidence and raises awareness, thereby providing a comprehensive approach to understand the association between RNA A-to-I editing and environmental exposures and between RNA A-to-I editing and diseases, mainly cancer. By using the publicly available data, it also aims to present the association between environmental exposures and expression RNA A-to-I editing genes, and to provide the association between RNA A-to-I editing genes and cancer.
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