Limits...
Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing.

Mazloomian A, Meyer IM - RNA Biol (2015)

Bottom Line: The majority of these editing events, however, cannot be associated with regulatory mechanisms.Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other.For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure.

View Article: PubMed Central - PubMed

Affiliation: a Centre for High-Throughput Biology; Department of Computer Science and Department of Medical Genetics ; University of British Columbia ; Vancouver ; BC , Canada.

ABSTRACT
RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10(-15)). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure.

No MeSH data available.


Related in: MedlinePlus

An example of a region where a conserved RNA secondary structure feature detected by investigating editing events can potentially influence alternative splicing. Rectangles at the bottom of the figure show exonic parts of Cip4 gene located on the reverse strand of the left arm of chromosome 3. The figure shows the structure predicted using RNAalifold in a region of 100 nucleotides around the splice site of an exonic region which is predicted to be alternatively used between tissues. Red arrows show predicted editing sites. Black arcs indicate alignment columns that are predicted to be base-paired, and black columns correspond to un-paired nucleotides. Green squares within the alignment show valid base-pairs and orange squares invalid base-pairs. Dark blue squares represent valid base-pairs with 2-sided mutations (compared to the most common base-pair in the pair of columns), probably in order to retain base-pairing potential. Likewise, light blue color represents single mutations to retain base-pairing potential. The existence of multiple compensatory mutations provides evidence for its functional importance throughout evolution. Figure generated using R-CHIE68
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4829317&req=5

f0005: An example of a region where a conserved RNA secondary structure feature detected by investigating editing events can potentially influence alternative splicing. Rectangles at the bottom of the figure show exonic parts of Cip4 gene located on the reverse strand of the left arm of chromosome 3. The figure shows the structure predicted using RNAalifold in a region of 100 nucleotides around the splice site of an exonic region which is predicted to be alternatively used between tissues. Red arrows show predicted editing sites. Black arcs indicate alignment columns that are predicted to be base-paired, and black columns correspond to un-paired nucleotides. Green squares within the alignment show valid base-pairs and orange squares invalid base-pairs. Dark blue squares represent valid base-pairs with 2-sided mutations (compared to the most common base-pair in the pair of columns), probably in order to retain base-pairing potential. Likewise, light blue color represents single mutations to retain base-pairing potential. The existence of multiple compensatory mutations provides evidence for its functional importance throughout evolution. Figure generated using R-CHIE68

Mentions: To discover potential mechanisms regulating the interplay between alternative splicing and RNA editing, we also searched for statistically significant conserved RNA secondary-structure features in the vicinity of exons where we found RNA editing and alternative splicing to co-occur. For this, we employed Transat51 on input alignments of 15 fly species downloaded from UCSC52 (We also added OregonR sequence to the alignment; see supplementary text for more details) around splice sites of alternatively spliced exonic parts where editing sites are also predicted (extended by 150 nucleotides on either side, a total of 167 regions). There already exist quite a few computational methods to predict evolutionarily conserved RNA secondary-structure.53-55 These programs, however, expect the input alignment to contain one more or less global secondary-structure, i.e., a structure spanning the entire alignment. As there a priori no reason to expect secondary-structure features relevant for RNA editing to involve the entire transcript – especially not longish fly pre-mRNAs in vivo – we use Transat as this program has been specifically designed to identify local, conserved RNA secondary-structure features such as the double-stranded regions needed for ADAR binding and RNA editing. Transat takes a set of aligned sequences and an evolutionary tree as input; potential helices in the alignment, and assigns a p-value to each of these helices. For 96 of the 167 regions (57%) where alternative splicing and RNA editing co-occur in our data we find one or more conserved RNA secondary-structure features (when we filter helices with p-value greater than 0.05 and helices shorter than 8 nucleotides). Figure 5 shows an example of these regions and the corresponding, conserved RNA secondary-structure detected by RNAalifold54 in this region. Multiple compensatory mutations for conserved base-pairs provide evolutionary evidence for a likely functional role of this double-stranded region. Finally, we applied RNAalifold to assess the stability of the global structures in these regions. Table S3 presents the list of the identified regions sorted by the energy of the global structure calculated by RNAalifold.Figure 5.


Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing.

Mazloomian A, Meyer IM - RNA Biol (2015)

An example of a region where a conserved RNA secondary structure feature detected by investigating editing events can potentially influence alternative splicing. Rectangles at the bottom of the figure show exonic parts of Cip4 gene located on the reverse strand of the left arm of chromosome 3. The figure shows the structure predicted using RNAalifold in a region of 100 nucleotides around the splice site of an exonic region which is predicted to be alternatively used between tissues. Red arrows show predicted editing sites. Black arcs indicate alignment columns that are predicted to be base-paired, and black columns correspond to un-paired nucleotides. Green squares within the alignment show valid base-pairs and orange squares invalid base-pairs. Dark blue squares represent valid base-pairs with 2-sided mutations (compared to the most common base-pair in the pair of columns), probably in order to retain base-pairing potential. Likewise, light blue color represents single mutations to retain base-pairing potential. The existence of multiple compensatory mutations provides evidence for its functional importance throughout evolution. Figure generated using R-CHIE68
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829317&req=5

f0005: An example of a region where a conserved RNA secondary structure feature detected by investigating editing events can potentially influence alternative splicing. Rectangles at the bottom of the figure show exonic parts of Cip4 gene located on the reverse strand of the left arm of chromosome 3. The figure shows the structure predicted using RNAalifold in a region of 100 nucleotides around the splice site of an exonic region which is predicted to be alternatively used between tissues. Red arrows show predicted editing sites. Black arcs indicate alignment columns that are predicted to be base-paired, and black columns correspond to un-paired nucleotides. Green squares within the alignment show valid base-pairs and orange squares invalid base-pairs. Dark blue squares represent valid base-pairs with 2-sided mutations (compared to the most common base-pair in the pair of columns), probably in order to retain base-pairing potential. Likewise, light blue color represents single mutations to retain base-pairing potential. The existence of multiple compensatory mutations provides evidence for its functional importance throughout evolution. Figure generated using R-CHIE68
Mentions: To discover potential mechanisms regulating the interplay between alternative splicing and RNA editing, we also searched for statistically significant conserved RNA secondary-structure features in the vicinity of exons where we found RNA editing and alternative splicing to co-occur. For this, we employed Transat51 on input alignments of 15 fly species downloaded from UCSC52 (We also added OregonR sequence to the alignment; see supplementary text for more details) around splice sites of alternatively spliced exonic parts where editing sites are also predicted (extended by 150 nucleotides on either side, a total of 167 regions). There already exist quite a few computational methods to predict evolutionarily conserved RNA secondary-structure.53-55 These programs, however, expect the input alignment to contain one more or less global secondary-structure, i.e., a structure spanning the entire alignment. As there a priori no reason to expect secondary-structure features relevant for RNA editing to involve the entire transcript – especially not longish fly pre-mRNAs in vivo – we use Transat as this program has been specifically designed to identify local, conserved RNA secondary-structure features such as the double-stranded regions needed for ADAR binding and RNA editing. Transat takes a set of aligned sequences and an evolutionary tree as input; potential helices in the alignment, and assigns a p-value to each of these helices. For 96 of the 167 regions (57%) where alternative splicing and RNA editing co-occur in our data we find one or more conserved RNA secondary-structure features (when we filter helices with p-value greater than 0.05 and helices shorter than 8 nucleotides). Figure 5 shows an example of these regions and the corresponding, conserved RNA secondary-structure detected by RNAalifold54 in this region. Multiple compensatory mutations for conserved base-pairs provide evolutionary evidence for a likely functional role of this double-stranded region. Finally, we applied RNAalifold to assess the stability of the global structures in these regions. Table S3 presents the list of the identified regions sorted by the energy of the global structure calculated by RNAalifold.Figure 5.

Bottom Line: The majority of these editing events, however, cannot be associated with regulatory mechanisms.Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other.For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure.

View Article: PubMed Central - PubMed

Affiliation: a Centre for High-Throughput Biology; Department of Computer Science and Department of Medical Genetics ; University of British Columbia ; Vancouver ; BC , Canada.

ABSTRACT
RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10(-15)). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure.

No MeSH data available.


Related in: MedlinePlus