Comparative Analysis Identifies Exonic Splicing Regulatory Sequences—The Complex Definition of Enhancers and Silencers

Goren, Amir; Ram, Oren; Amit, Maayan; Keren, Hadas; Lev-Maor, Galit; Vig, Ida; Pupko, Tal; Ast, Gil

doi:10.1016/j.molcel.2006.05.008

Cited by 282 publications

(321 citation statements)

References 53 publications

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“…This sequence is not present in the G6PD regulatory element. The G6PD regulatory sequence does score highly as a potential exonic regulatory sequence using a new algorithm developed using computational analysis of 46,103 exons [45]. This is consistent with our most recent data demonstrating that the region from nt 43-72 of exon 12 is an ESS [46].…”

Section: Discussionsupporting

confidence: 85%

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

Griffith¹,

Walsh²,

Szeszel-Fedorowicz³

et al. 2006

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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SummaryNutrient regulation of glucose-6-phosphate dehydrogenase (G6PD) expression occurs through changes in the rate of splicing of G6PD pre-mRNA. This posttranscriptional mechanism accounts for the 12-to 15-fold increase in G6PD expression in livers of mice that were starved and then refed a high-carbohydrate diet. Regulation of G6PD pre-mRNA splicing requires a cis-acting element in exon 12 of the pre-mRNA. Using RNA probes to exon 12 and nuclear extracts from livers of mice that were starved or refed, proteins of 60 kDa and 37 kDa were detected bound to nucleotides 65-79 of exon 12 and this binding was decreased by 50% with nuclear extracts from refed mice. The proteins were identified as hnRNP K, and L, and hnRNP A2/B1 by LC-MS/MS. The decrease in binding of these proteins to exon 12 during refeeding was not accompanied by a decrease in the total amount of these proteins in total nuclear extract. HnRNPs K, L and A2/B1 have known roles in the regulation of mRNA splicing. The decrease in binding of these proteins during treatments that increase G6PD expression is consistent with a role for these proteins in the inhibition of G6PD mRNA splicing.

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Section: Discussionsupporting

confidence: 85%

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

Griffith¹,

Walsh²,

Szeszel-Fedorowicz³

et al. 2006

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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“…In particular, the ESEs and ESSs exhibited opposite evolutionary trends, consistent with their interpretation as elements that act with a considerable degree of autonomy. This contrary behavior would not be expected if ESEs and ESSs are considered to be facultative motifs entirely dependent on context ("ESRs," as proposed by Goren et al 2006). This is not to say that this autonomy is complete, as there is experimental evidence that context can indeed determine the action of a splicing motif (e.g., Kanopka et al 1996;Goren et al 2006).…”

Section: Predicted Ese and Ess Motifs Exhibit Evolutionary Behavior Cmentioning

confidence: 93%

“…This contrary behavior would not be expected if ESEs and ESSs are considered to be facultative motifs entirely dependent on context ("ESRs," as proposed by Goren et al 2006). This is not to say that this autonomy is complete, as there is experimental evidence that context can indeed determine the action of a splicing motif (e.g., Kanopka et al 1996;Goren et al 2006). Rather, we conclude that ESEs and ESSs usually and overall act according to their designation.…”

Section: Predicted Ese and Ess Motifs Exhibit Evolutionary Behavior Cmentioning

confidence: 93%

“…Similarly, we do not yet understand the possible combinatorial rules for ESE and ESS interaction. An ESE can act as an ESS and vice versa depending on position (Kanopka et al 1996;Goren et al 2006). Finally, compensatory changes may be acting via changes in intronic motifs flanking these exons (Zhang et al , 2005cYeo et al 2005); we did not search for intronic changes here in the absence of validated global sets of such motifs.…”

Section: Genome Research 537mentioning

confidence: 99%

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Positive selection acting on splicing motifs reflects compensatory evolution

Ke¹,

Zhang²,

Chasin³

2008

Genome Res.

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We have used comparative genomics to characterize the evolutionary behavior of predicted splicing regulatory motifs. Using base substitution rates in intronic regions as a calibrator for neutral change, we found a strong avoidance of synonymous substitutions that disrupt predicted exonic splicing enhancers or create predicted exonic splicing silencers. These results attest to the functionality of the hexameric motif set used and suggest that they are subject to purifying selection. We also found that synonymous substitutions in constitutive exons tend to create exonic splicing enhancers and to disrupt exonic splicing silencers, implying positive selection for these splicing promoting events. We present evidence that this positive selection is the result of splicing-positive events compensating for splicing-negative events as well as for mutations that weaken splice-site sequences. Such compensatory events include nonsynonymous mutations, synonymous mutations, and mutations at splice sites. Compensation was also seen from the fact that orthologous exons tend to maintain the same number of predicted splicing motifs. Our data fit a splicing compensation model of exon evolution, in which selection for splicing-positive mutations takes place to counter the effect of an ongoing splicing-negative mutational process, with the exon as a whole being conserved as a unit of splicing. In the course of this analysis, we observed that synonymous positions in general are conserved relative to intronic sequences, suggesting that messenger RNA molecules are rich in sequence information for functions beyond protein coding and splicing.[Supplemental material is available online at www.genome.org.]In addition to sequences that specify their cognate polypeptides, the open reading frames of eukaryotic messenger RNAs (mRNAs) are likely to contain additional information governing the functioning of these molecules; these functions include exon splicing and mRNA transport, localization, stability, and translation. If so, the evolution of mRNA sequence would be constrained by purifying selection against the loss of this information. This selection should be evident by examining the rate and quality of base substitutions that do not alter protein coding (synonymous substitutions) but may alter one of the abovementioned functions.Mutation rates in open reading frames have been extensively characterized by two parameters: K a , the rate of mutations that result in amino acid substitutions; and K s , the rate at synonymous sites. The latter has most often been assumed to reflect neutral change and has been used to normalize the mutation rates in a given gene, with the K a /K s ratio serving as an inverse measure of protein sequence conservation (Hurst 2002). More recently, the neutrality of K s has been challenged, with accumulating evidence for selection acting at synonymous sites (for reviews, see Chamary et al. 2006;Xing and Lee 2006). Some of this evidence has been based on comparisons between mutation rates in predicted exonic splicing e...

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“…18 Indeed, the nucleotide change at position 427 in hsvtk2 generates a hexamer CTCATG that was recently identified as a putative exonic splicing regulatory sequence by bioinformatic analysis. 19 Further, by running this sequence in the ESEfinder, we find that the nucleotide change from A to G creates a binding site for SR proteins (SF2/ASF, SRp40 and SRp55) that does not exist in hsvtk1. 20 Alternatively, these minute sequence changes may abrogate an intron splicing silencer in hsvtk2 otherwise present in hsvtk1 and 3.…”

Section: Discussionmentioning

confidence: 94%

Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes

et al. 2008

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The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that g-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovirmediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in g-retroviral or lentiviral vectors.

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Comparative Analysis Identifies Exonic Splicing Regulatory Sequences—The Complex Definition of Enhancers and Silencers

Cited by 282 publications

References 53 publications

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

Positive selection acting on splicing motifs reflects compensatory evolution

Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes

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