Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery

Schwartz, Schraga; Gal-Mark, Nurit; Kfir, Nir; Ram, Oren; Kim, Eddo; Ast, Gil

doi:10.1371/journal.pcbi.1000300

Cited by 57 publications

(67 citation statements)

References 57 publications

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“…A distant cluster of editing sites can potentially alter the splicing pattern of a remote region by changing the structure and stability of RNA. Indeed, a growing body of evidence suggests that AS is also controlled by the secondary structure of RNA (Shepard and Hertel 2008;Schwartz et al 2009a;Pervouchine et al 2011). A probable example for this regulation in our analysis is the third exon of SEPN1, where two reverse-oriented Alus form the substrate for both editing and splicing (Supplement 1, Supplemental Fig.…”

Section: Discussionmentioning

confidence: 79%

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Solomon

Oren

Safran

et al. 2013

RNA

109

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Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNAseq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

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Section: Discussionmentioning

confidence: 79%

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Solomon

Oren

Safran

et al. 2013

RNA

109

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“…Constitutive and alternative exon data set construction Data sets for human constitutive and alternative exons were obtained as in Schwartz et al (2009a), based on the RefSeq and the spliced EST tracks of the human genome from the UCSC Genome Browser (http://genome.ucsc.edu/; Karolchik et al 2003). Constitutive exons were defined as those having 100% inclusion supported by at least five ESTs, yielding a total of 111,617 constitutive exons.…”

Section: Methodsmentioning

confidence: 99%

“…We retrieved a total of 4433 human alternative cassette exons and 111,617 constitutive exons based on the RefSeq tracks from the UCSC Genome Browser (http://genome.ucsc.edu/; Karolchik et al 2003) as was previously published (Schwartz et al 2009a). Classifications of exons into alternative and constitutive were based on human genome EST support alone (see Methods).…”

Section: Constitutive and Alternative Conservation Analysismentioning

confidence: 99%

Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

Gelfman¹,

Burstein²,

Penn³

et al. 2011

Genome Res.

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Exon-intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon-intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 39 and 59 splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery.[Supplemental material is available for this article.]In the process of splicing, introns are removed from an mRNA precursor (pre-mRNA), and exons are ligated to form a mature mRNA (Black 2003). Exons and introns are recognized in the splicing process by many different signals and interactions along the exonintron structure. Several signals along the pre-mRNA help the splicing machinery to recognize exon-intron junctions: The 39 and 59 splice sites (39ss and 59ss) located on both exon-intron junctions, and the branch site and polypyrimidine tract (PPT) located upstream of the 39ss (Black 2003). In alternative splicing, the splicing mechanism produces more than one mRNA from a single premRNA (Graveley 2001). This is done by the splicing of different sets of exons from a single pre-mRNA, resulting in an increased number of protein isoforms that can be synthesized from one gene. Previous studies revealed that the percentage of exons undergoing alternative splicing is higher in vertebrates compared with invertebrates, and in human compared with other vertebrates ). This suggests that alternative splicing has a major role in the production of higher levels of biological complexity.Exon-intron structure plays a major role in the recognition of exons by the splicing machinery. It was previously demon...

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“…However, it may be the secondary structure of the 5S rRNAlike element that actually influences the splicing decision. There is evidence that secondary structure can influence alternative splicing 27,28 potentially by masking splice sites. 28 Schwartz et al 27 determined that secondary structure plays a role in would interact with its own pre-mRNA via the 5S rRNA-like structural elements helix IV and loop E. This would influence inclusion of the PTC containing exon and trigger NMD.…”

Section: W Brad Barbazukmentioning

confidence: 99%

“…There is evidence that secondary structure can influence alternative splicing 27,28 potentially by masking splice sites. 28 Schwartz et al 27 determined that secondary structure plays a role in would interact with its own pre-mRNA via the 5S rRNA-like structural elements helix IV and loop E. This would influence inclusion of the PTC containing exon and trigger NMD. Conversely, when TFIIIA levels are low, the PTC containing exon is skipped during mRNA processing so that the ES isoform is produced, which leads to an increase in TFIIIA protein levels.…”

Section: W Brad Barbazukmentioning

confidence: 99%

A conserved alternative splicing event in plants reveals an ancient exonization of 5S rRNA that regulates TFIIIA

Barbazuk

2010

RNA Biology

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Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery

Cited by 57 publications

References 57 publications

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

A conserved alternative splicing event in plants reveals an ancient exonization of 5S rRNA that regulates TFIIIA

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