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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cell. Author manuscript; available in PMC 2013 January 13.
Published in final edited form as:
PMCID: PMC3409466
NIHMSID: NIHMS394924

NASCENT-SEQ INDICATES WIDESPREAD COTRANSCRIPTIONAL RNA EDITING IN DROSOPHILA

SUMMARY

The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila, by isolating nascent RNA from adult fly heads and subjecting samples to high throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR null mutant strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally.

Keywords: nascent sequencing, cotranscriptional pre-mRNA editing, splicing, ADAR

INTRODUCTION

RNA editing increases the informational content of the genome. Moreover, independent experiments in different systems indicate that editing is essential for biological function. Although Drosophila null for ADAR are morphologically normal with no life span defect, they exhibit severe behavioral and locomotor defects (Palladino et al., 2000). They probably reflect the failure to edit transcripts encoding many key proteins involved in fast electrical and chemical neurotransmission, e.g., shab (Ryan et al., 2008) and shaker (Ingleby et al., 2009). The effects of mRNA editing in mammals are also striking. ADAR1 and ADAR2 are both essential in mice; ADAR1−/− mice die as early embryos, and ADAR2 −/− mice die post-natally of juvenile onset epilepsy-like symptoms (Hartner et al., 2004; Higuchi et al., 2000; Wang et al., 2000; Wang et al., 2004). For example, the transcript encoding the GluR-B subunit of the AMPA receptor is an ADAR-substrate. Even mice heterozygous for editing-incompetent GluR-B-engineered alleles (unedited/edited) do not survive (Brusa et al., 1995; Feldmeyer et al., 1999).

Editing has been shown to occur primarily within the pre-mRNA of target genes (Higuchi et al., 1993). Although the rules that define an ADAR substrate are still uncertain (Eggington et al., 2011), the introns of several well studied examples contain functional elements termed editing site complementary sequences (ECS). Intra-molecular base pairing between the ECS and the target exon creates a recognition element for ADAR binding. Based on RNase-sensitivity, there is evidence that ADAR is recruited to nascent RNA, at specific chromosomal loci (Das et al., 2007; Eckmann and Jantsch, 1999; Sallacz and Jantsch, 2005). However, the extent to which the covalent editing events occur on nascent RNA, i.e., prior to 3′ end formation, has not been broadly investigated.

RESULTS

Cotranscriptional editing of pre-mRNA

To assess the extent of cotranscriptional editing in the Drosophila system, we isolated nascent RNA from adult fly heads and subjected 12 samples (2 datasets each containing 6 samples; see Experimental Procedures) to high throughput sequencing using a standard Illumina single end protocol. As expected, the data showed that nascent RNA contains significant levels of intron signal compared to cytoplasmic mRNA and to total mRNA (Fig. S1A, S1B for an example of Nascent-seq vs pA-seq). To aid in filtering out possible SNPS, we also sequenced DNA from the wildtype yw strain to 39X coverage and only considered sites that did not contain a single G in the DNA. After mapping the RNA sequences to the reference genome, we searched for the occurrence of 1 or more guanosines at each site in our 12 samples. Our most stringent criterion required a G in at least 10 of the 12 samples (and 5 of 6 samples within each dataset) as well as zero Gs in the genomic sequence (Fig. 1A). Using a log likelihood score, we classified sites into high or low ranking (see Experimental Procedures for more details).

Figure 1
Editing of mRNA edited sites is cotranscriptional.

The nascent RNA contains 621 high ranking edited sites within RefSeq annotated exons indicating that cotranscriptional editing is widespread (Fig. 1A; Table S1). Additionally, there are another 251 lower ranking sites that did not meet the most stringent thresholds. To address data reproducibility, we pooled the 6 samples from each dataset and calculated the average percent editing level at every edited site within the individual sets; there was remarkably high reproducibility (R=0.96; Fig. 1B). Most sites also show a rather low percentage of nascent editing; peaking at 15% (Fig. 1C). However, 22% of the sites are edited over 50%, i.e.., G>A.

To compare nascent editing levels with more standard mRNA data, we sequenced pA RNA from two independent samples of yw fly heads (see Experimental Procedures). These samples were from the same batch of fly heads used for the Nascent-seq data above. We also used the same analysis pipeline to identify editing sites in the mRNA data. However, since we only sequenced two mRNA samples, we required in addition that mRNA edited sites be observed in both samples.

The mRNA data indicate 276 high ranking edited sites (Table S1). Significantly, 93% of these are present in the Nascent-seq, indicating they are cotranscriptionally edited (Fig. 1D); only 19 mRNA sites are not in these high ranking nascent sites. In contrast, 41% of the high ranking nascent sites were found in the mRNA data. We suspect that many of these sites are absent because of the rather low coverage, because only two samples were sequenced. Indeed, over 92% of the nascent sites are found in the mRNA data if the site is only required in 1 of the 2 mRNA preps (see below).

mRNA editing level is determined cotranscriptionally

The 93% overlap between edited sites in mRNA and in nascent RNA allowed us to directly compare their editing levels. Although mRNA editing levels are slightly higher than nascent editing levels (8% on average; p = 1e-21), the two are highly correlated (R=0.83; Fig. 2A). A similar high correlation of nascent editing levels is also observed with 12 pA-seq samples from another wildtype strain, Canton-S (R=0.90; Fig. 2B), indicating that the level of editing is set cotranscriptionally.

Figure 2
mRNA editing level is set cotranscriptionally.

We then compared the rankings of exon base pair coverage, a measure of relative RNA abundance between the nascent and mRNA datasets (Fig. 2C). This was done by determining the log ratio of nascent read signal rankings divided by mRNA read signal rankings for edited genes compared to all genes. Edited genes have a lower relative ranking in mRNA compared to nascent RNA (Fig. 2C; p = 1e-81). The simplest interpretation is that edited mRNAs have a shorter half-life than non-edited mRNAs. Alternatively editing affects transcription and causes a high nascent signal. Because longer mRNAs have lower mRNA/nascent RNA read ratios than shorter mRNAs (data not shown), we generated the same log ratios as a function of transcript length. The same lower relative mRNA ranking was observed even within restricted mRNA length distributions (Fig. 2D, S2A, S2B). We note that the distinction between edited and non-edited genes does not seem to be solely a characteristic of neuronal genes (data not shown).

Intron editing is widespread

Editing within introns containing ECS sequences, has been previously described for the mouse GluR-B genes (Higuchi et al., 1993; Rueter et al., 1995). Because our nascent RNA data are highly enriched for intron signal (Fig. S1A, S1B), we searched for genome-wide intron editing within RefSeq annotated introns. There were 729 high ranking sites and an additional 171 low ranking sites, indicating a surprisingly high level of nascent intron editing. This makes a total of 1350 high ranking nascent intron plus exon sites, with only a small fraction of the latter located within 5′ and 3′ UTRs (Fig. 3A; Table S1). The percentage of editing sites is slightly biased toward coding regions (36%; Fig. 3A) relative to the percentage of coding nucleotides in the genome (27%); this suggests some positive selection for coding region editing or negative selection for editing elsewhere.

Figure 3
A majority of edited sites occurs within introns.

Specific or multiple editing, where one or only a few sites within a RNA are edited, is believed to occur on short imperfectly paired dsRNA; promiscuous editing, i.e., many edited sites, occurs on long perfectly dsRNA regions (DeCerbo and Carmichael, 2005b). We observed that intron editing and exon editing have similar distributions of specific, multiple and promiscuous editing (Fig. 3B). This suggests that intron editing follows the same rules as exon editing (see below).

Based on gene annotation data, we grouped nascent edited genes into three classes (Fig. 3C). The first class, genes edited within introns as well as exons, contains 58 genes and represents 15% of all edited genes. This co-occurrence of intron editing and exon editing is much greater than expected for independent events (p = 1e-10), indicating that intron editing and exon editing are linked.

To determine if exon and intron edited events occur within the same nascent transcripts in this first class of editing genes, we PCR amplified and cloned regions of 3 genes containing both intron and exon editing (see Experimental Procedures). We then performed Sanger sequencing from both ends of individual clones. Exon and intron editing indeed occur within the same transcripts in all 3 genes (Fig. 3D, S3A). Over 66% of these exon-edited transcripts contain at least one edited intron site, and over 79% of intron-edited transcripts contain at least one edited exon site. Furthermore, editing levels calculated from the Sanger sequencing data are remarkably well-correlated with the levels determined from the Nascent-seq data (R=0.98 exon sites, R=0.77 intron sites; Fig. S3B).

Shaker, a well studied potassium channel, is a good example of this first class. It has 6 previously identified exon sites (Hoopengardner et al., 2003; Ingleby et al., 2009), and the nascent data contain all 6 sites and no others. However, the nascent data also identify 52 intron-edited sites within this gene (Fig. 3E). They are all distributed throughout the 3′ half of the gene locus.

The second class, editing within introns only, accounts for 36% of all edited genes (Fig. 3C). rdgA, a gene involved in vision, is an example and has 26 intron-edited sites (Fig. 3F). They appear uniformly distributed across the transcription unit.

The third class contains genes edited in exons only and accounts for almost half of all edited genes (49%; Fig. 3C). An example is CG10077 with 9 sites distributed between the 5′ and 3′ exons (Fig. 3G). 5 of these sites were recently identified by the modENCODE project (Graveley et al., 2011). Because this consortium generated a deep mRNA dataset by sequencing mRNA from a diverse set of developmental tissues including whole adults (Graveley et al., 2011), we were surprised that the nascent data identified all 5 of their CG10077 sites as well as 4 more.

Most identified mRNA sites are cotranscriptionally edited

To facilitate a more comprehensive comparison with the modENCODE data, we first removed modENCODE sites with one or more Gs and lower than 10x coverage in our genomic sequencing data. Of the 665 remaining modENCODE sites, our high ranking nascent head data identified 47% of them, 51% including lower ranking sites, and up to 63% when only requiring the presence of a site in 6 of 12 nascent samples (Fig. 4A). Conversely, 49% of the nascent sites are absent from the extensive modENCODE dataset. Because a much larger fraction of the nascent sites are present in our yw head mRNA samples despite lower coverage, one simple explanation is differences in tissue sources. If much of Drosophila editing is from the nervous system, then the choice of diverse developmental stages and tissues by the modENCODE project might have underemphasized a key source of editing.

Figure 4
Comparison of previously identified mRNA sites with cotranscriptionally edited sites.

In addition to the modENCODE dataset (Graveley et al., 2011), there is another experimentally verified list of edited mRNA sites (Hoopengardner et al., 2003; Jepson et al., 2011). The nascent data contain 81% of them; 92% when considering sites edited in 6 of 12 samples (Fig. 4B). For example, the nascent data identify 10 edited sites within exons of the acetylcholine receptor subunit nAcRalpha-34E (dalpha5) (Fig. 4C). Of these 10, 6 were identical to the 8 edited sites previously identified within this gene (Grauso et al., 2002; Hoopengardner et al., 2003; Jepson et al., 2011). We conclude that at least 75% of these previously identified sites are cotranscriptionally edited, consistent with the rest of the data indicating that most editing occurs cotranscriptionally.

The two extra sites present only in the nascent data lie within an exon that contributes to the ligand-binding domain of nAcRalpha-34E; it is also an alternatively spliced exon (Fig. 4C, 4D) (Grauso et al., 2002). Interestingly, the nascent sequencing data also suggest that the intron surrounding this exon has a lower cotranscriptional splicing efficiency, i.e., a higher level of intron retention in nascent RNA than average introns in the nascent data (Fig. 4D). Although these two sites could be absent from and not relevant to mature mRNA and therefore to coding potential, alternative splicing might make some edited sites more difficult to detect in mRNA than in nascent RNA.

Most exon as well as intron sites are not edited in ADAR mutant flies

To validate the identified nascent exon and intron edited sites and also to control for background noise, we sequenced nascent RNA from an ADAR mutant fly strain, ADAR0. It has severely compromised ADAR activity (see Experimental Procedures). Given that we obtained very few viable ADAR0 flies, we modified our standard nascent RNA protocol to accommodate fewer fly heads and also sequenced the same number of yw and wild-type FM7A control fly heads. We prepared libraries from two replicates of the three strains and examined the 1350 exon and intron sites. Only sites that occurred in both replicates of the yw and FM7 controls were considered, which resulted in 609 editing events: 374 exon sites and 235 intron sites.

The editing levels of almost all exon and intron sites were dramatically reduced in ADAR mutant flies (Fig. 5A, 5B). We used these data to calculate false positive rates for exon- and intron-editing, which were 4.5% and 5.1%, respectively. We conclude that the vast majority of newly identified sites are real and due to ADAR activity.

Figure 5
Most exon and intron sites are poorly edited in ADAR0 mutant flies.

Intron editing occurs within the syt1 ECS and is evolutionarily conserved

Some ADAR substrates are formed when the target exon forms a double stranded structure with a complementary sequence (ECS) in the intron (Egebjerg et al., 1994; Hanrahan et al., 2000; Herb et al., 1996; Higuchi et al., 1993; Reenan, 2005). Moreover an intron containing the ECS in the transcript encoding the GluR-B is edited (Higuchi et al., 1993; Rueter et al., 1995). To gain further insight into intron editing, we focused on two syt1 ECS regions that are exceptionally well-characterized for syt1 exon editing (Reenan, 2005). These two regions are located within the subsequent intron and are necessary for formation of the ADAR substrate and for exon editing (Fig. 6A). Mismatches introduced into these regions abolish editing within the exon, and can be rescued with compensatory mutations that restore pairing (Reenan, 2005). We searched for editing within this intron and found 4 intronic sites. All 4 are located within the two ECS regions and directly opposite each other in the dsRNA structure (Fig. 6A).

Figure 6
syt1 intron editing is conserved and a link between editing and splicing.

We next asked whether all intron-edited sites including the sites in syt1were evolutionary conserved across different Drosophila species. We first addressed the issue computationally and did not observe significant conservation of edited intron adenosines compared to all intron nucleotides sequences (data not shown). We then addressed the issue experimentally, by selecting 2 syt1 intronic sites (I069 and I071 sites; Fig. 6A) and assessing intron editing by Sanger sequencing PCR-amplified cDNA and genomic DNA fragments across 4 Drosophila species (including the distantly related D. mojavensis). Editing of both sites was apparent in all 4 species (Fig. 6B), indicating they have been evolutionary conserved over 63 million years (Tamura et al., 2004).

Relationship between editing and splicing

Given that a 5′ splice site is contained within the syt1 pseudoknot structure (Reenan, 2005), we considered that splicing may compete with editing. To test this hypothesis, we compared the Nascent-seq signal within this intron between ADAR0 and wildtype controls. Significantly higher levels of intron signal were present in the ADAR0 strain compared to both yw and background genotype controls (Fig. 6C), suggesting that ADAR activity increases syt1 cotranscriptional splicing. This increased intron retention (1.55-fold increase, Fig. 6D) was confirmed by qRT-PCR of total RNA (2.76-fold increase, p < 0.05, Fig. 6E).

The connection between RNA editing and splicing of syt1 inspired us to address this relationship genome-wide. To this end, we used several metrics that reflect splicing, including intron retention; it measures the sequencing signal within each intron normalized to the exon signal across each gene (Khodor et al., 2011). Edited introns have significantly higher intron retention compared to all introns (median of 0.468 compared to 0.268, respectively; p < 1e-15; Fig. 6F, S4A, S4B). Moreover, the introns surrounding exon-edited only genes exhibit significantly less intron signal (median upstream intron: 0.21, median downstream intron: 0.185; p < 1e-5), when compared to the population of introns (median of 0.268). We interpret these results to indicate that intron editing occurs preferentially on introns that are poorly cotranscriptionally spliced. Because there is no global change in intron retention of intron-edited genes in ADAR0 mutant flies (Fig. S4C), either another editing component inhibits cotranscriptional splicing or the poor cotranscriptional splicing is upstream of editing, perhaps to facilitate ADAR recognition or function.

DISCUSSION

The data shown here indicate that Nascent-seq is an efficient and comprehensive strategy to identify editing sites. Although a minor contribution by post-transcriptional editing could account for the slight increase (8%) in mRNA editing levels compared to nascent RNA (Fig. 2A), most A-to-I editing occurs cotranscriptionally. Moreover it requires ADAR, as indicated by sequencing of the ADAR-null strain. In addition, this genome-wide study of A-to-I conversion revealed a surprisingly large number of intronic sites. A paper published during revision of this manuscript also reported extensive intron editing in the human system (Peng et al., 2012). The nascent RNA approach used here also suggests mechanistic links between editing and cotranscriptional splicing as well as between intron editing and exon editing.

For example, the statistically significant overlap of intron and exon editing on the same genes suggests that they occur on the same transcripts, which is confirmed by the Sanger sequencing results (Fig. 3D). Taken together with the dramatic reduction of intron as well as exon editing in the ADAR0 strain, we suggest that they are mechanistically similar and in some cases linked, e.g., the same ADAR-containing complex could catalyze intron as well as exon editing within one transcript. Given the possibility of unannotated alternate splicing (Fig. 4D), intron editing may in some cases function similarly to exon editing and expand coding potential.

Because introns and their ECS regions have been shown to play an integral role in defining most well-studied ADAR substrates, one can imagine that intron editing facilitates or inhibits exon editing. A positive or regulatory role of intron editing on exon editing is possible in the case of the 15% of edited genes with editing events within introns as well as exons. Regulatory interactions might occur only indirectly, i.e., without ECS editing. However, a direct effect of ECS editing on exon editing is plausible in the case of syt1 with 4 intron-edited sites within the known ECS (Fig. 6A).

In contrast to the genes with both intron and exon editing, almost half of all edited genes only had exon editing, indicating that it is biased towards exons and likely under positive selection. Most Drosophila introns are underrepresented in nascent RNA (Fig. 6F), indicating that they are efficiently spliced cotranscriptionally (Khodor et al., 2011). This might cause an underestimate of intron editing frequency, which could also be related to the slight but significantly lower intron retention observed in exon-only edited genes (Fig. 6F). However, more than 75% of “exon-only edited genes” are still missing intron editing when the thresholds for identifying editing are reduced from 10 of 12 samples to 6 of 12 samples (data not shown). We cannot exclude the possibility that deeper sequencing would alter some of these conclusions, quantitatively or perhaps even qualitatively.

The introns of exon-only edited genes may still contain functional ECS regions, or these exons might form ADAR substrates autonomously. This is the case for an editing site that is recognized by ADAR without an intron (Keegan et al., 2005) and for the transcript of the intronless gene Kv1.1 (Bhalla et al., 2004). These examples suggest that even mature transcripts have the potentially to be edited. Another alternative is that some exon-only edited genes use trans-acting RNAs to form ds RNA regions.

There are two extreme models for the relationship between editing and splicing: splicing occurs before editing, or editing occurs before splicing. If splicing is first, intron editing could be negatively impacted because potential intron substrate regions might be removed from nascent RNA before intron editing can occur. This raises an apparent paradox: might splicing also inhibit exon editing by removing a required ECS from pre-mRNA prior to editing? If editing is first, might it inhibit splicing (Bratt and Ohman, 2003; Laurencikiene et al., 2006)? This possibility is supported by the finding that the introns surrounding the nAcRalpha-34E alternative exon containing the two extra edited sites are inefficiently spliced (Fig. 4D). Also consistent with this notion is the fact that edited introns on average have significantly more intron retention than all introns (Fig. 6F). Alternatively, this might just reflect inefficient cotranscriptional splicing, which is mechanistically unrelated to editing. However, the inefficient splicing might still help promote editing.

Exon editing might even promote splicing. In the case of exon-only edited genes, this might help remove introns before less efficient intron editing events have a chance to occur. It is interesting in this context that numerous ADAR substrate pre-mRNAs sequester splicing signals within editing-relevant dsRNA structures (Burns et al., 1997; Hanrahan et al., 2000; Higuchi et al., 1993; Reenan, 2005). For example, the syt1 intron forms a pseudoknot structure which contains the 5′ splice site (Reenan, 2005). Moreover, there is a significant increase in intron retention of the syt1 intron in ADAR0 nascent RNA compared to WT controls (Fig. 6C, 6D, 6E). Editing within the syt1 DI and DII dsRNA may therefore destabilize these structures (Bass and Weintraub, 1988; Serra et al., 2004) and allow splicing to proceed.

However, there is no general effect on the retention of edited introns in ADAR0 nascent RNA compared to WT controls, e.g., the higher intron retention of edited introns may be upstream of editing. The syt1 edited intron therefore appears to be an exception. There are even a small number of edited introns that respond in the opposite way, namely, a decrease in intron retention in the ADAR nascent RNA compared to control nascent RNA (data not shown).

Nonetheless, there is good evidence in the literature for a regulatory interface between splicing and editing (Rieder and Reenan, 2011). For example, an intron editing site has been reported to convert a cryptic/benign splice site into a favored splicing acceptor; it induces an alternative splicing event within an ADAR2 transcript (Dawson et al., 2004). There is other evidence that editing promotes splicing: ADAR2−/− mice not only have significantly less edited GluR-B pre-mRNA but also accumulate pre-mRNA; they also have 5-fold less mRNA than wild-type (Higuchi 2000). Based on the fact that Drosophila editing levels are set cotranscriptionally (Fig. 2A, 2B) and that most splicing occurs cotranscriptionally (Khodor et al., 2011), we favor the notion that some intron-only editing impacts pre-mRNA splicing. Alternatively or in addition, cotranscriptional intron editing may impact other aspects of nuclear RNA processing as suggested by its effect on transcript retention within nuclei (DeCerbo and Carmichael, 2005a; Zhang and Carmichael, 2001).

EXPERIMENTAL PROCEDURES

Fly Strains

ADAR0/FM7a flies were obtained from the Reenan lab, and were generated as a result of previous work (Jepson et al., 2011). This strain was generated from homologous recombination events at the ADAR locus. And although the extent of the deletion is unknown, at least the catalytic domain is deleted. D.simulans (14021-0251.194), D.yakuba (14021 0261.01), D. pseudoobscura (14011-0121.94), D.mojavensis (15081-1352.22) fly strains were obtained from the Griffith Lab, but originate from the Drosophila Species Stock Center (DSSC) at the University of California, San Diego. D.melanogaster yellow white (yw) and Canton-S (Cs) lab stock flies were used.

Nascent RNA Extraction

yw flies were entrained to three days of 12hr:12hr light and dark cycles at 25 degrees. Flies were collected at 4 hour intervals on the 4th day and frozen on dry ice. Fly heads were then isolated with brass sieves. Nascent RNA was extracted as described (Khodor et al., 2011). Briefly, purified nuclei were isolated from 1 ml of fly heads for each sample, and resuspended in 1 ml nuclear lysis buffer. An equal volume of 2X NUN (NaCl, Urea, NP40) buffer was added to each sample and incubated on ice for 20 minutes. The nascent RNA containing complex was pelleted. Nascent RNA was extracted from the NUN pellet with Invitrogen TRIzol Reagent using the manufacturer’s protocol.

Total RNA Extraction

Total RNA was extracted with TRIzol Reagent (Invitrogen) using the manufacturer’s protocol.

Library Construction and Sequencing

Sequencing library construction was performed using standard Illumina protocol as previously described (Khodor et al., 2011)(see Supplemental Information).

Genomic DNA Extraction

Genomic DNA was extracted using standard protocol with minor modifications (see Supplemental Information).

PCR and qRT-PCR

PCR and RTPCR were performed with Platinum Taq (Invitrogen) and Syber Master Mix (Qiagen) respectively, using standard protocols as described (Abruzzi et al., 2011). qRT-PCR from ADAR0 and FM7a cDNA samples were performed from 3 independent replicates each. T-tests with unequal variances were used to test significance between the samples. The sequences of the primers used are supplied in Supplemental Table 3.

PCR Purification and Sanger Sequencing

PCR amplicons were gel purified and Sanger sequenced by Genewiz, Inc.. 2 replicates of cDNA made from 2 independent samples of total RNA were used for the I069 and I071 intron validation across species. 31-39 plasmids from the pGEM-T cloning were sequenced for each cloned amplicon, from both T7 and SP6 ends.

pGEM-T Cloning

PCR amplicons from cDNA made from an independent sample of Nascent RNA were gel purified and cloned into pGEM-T (Promega) using manufacturers protocol. Cloned amplicons were transfected into competent cells and plated on LB 100/XGAL media. White colonies were selected, grown in liquid media and plasmids were extracted with a miniprep kit (Qiagen).

Sequencing Alignment

Nascent RNA, yw mRNA and yw genomic libraries were aligned to the dm3 Drosophila melanogaster genome with Tophat (Trapnell et al., 2009) using the parameters “ -m 1 -F 0 --microexon-search --no-closure-search -G exon20110421.gtf --solexa1.3-quals -I 50000 ” Cs paired end samples were aligned with Tophat using the parameters “-r 50 -m 1 -F 0 - I 50000 -g 1 -G exon20110421.gtf”, and reads with overlapping ends were removed and remapped. Cs single end samples were aligned with Tophat using the parameters “-m 1 -F 0 -g 1 --microexon-search --no-closure-search -G exon20110421.gtf --solexa1.3-quals -I 50000”. We used annotation from UCSC Genes and Gene Prediction group, flybaseGene Track/table Apr. 2006 assembly to aid Tophat in the alignment of splice junctions (exon20110421.gtf).

RNA Editing Site Identification

Base frequencies were calculated within exons and introns of UCSC annotation. Genes with multiple isoforms were flattened, where overlapping exons generate one exon. Base positions with one or more Gs in the nascent datasets and zero Gs in the sequenced genomic DNA were identified. We required that editing sites occur in at least 5 of 6 samples within each set of replicate time points, for a total of 10 of 12 independent occurrences for each site. To avoid potential mismapping of reads at splice junctions by Tophat, we required that edited sites occur in at least one of the two middle quadrants of at least one read. Intronic sites that occurred within 10 bases of an annotated splice site were also discarded. We also ranked our datasets by using the following g-test log likelihood metric.

a=EditedBases b=TotalBases c=GenomicEditBases d=GenomicTotalBases

log likelihood metric (logl) = 2*a*ln(a/EditExp) + 2*c*ln(c/GExp)

EditExp = b*(a+c)/(b+d);

GExp = d*(a+c)/(b+d);

When considering only zero G bases in the genomic, the equation simplifies to: logl = 2*a*log(a/EditExp)

This metric was calculated for each sample and summed over all 12 replicates with 12 degrees of freedom. A one sided chi squared p-value was generated and used to separate editing sites into high ranking with a 1e-6 cut-off and low ranking with a 0.05 cut-off. We applied a similar approach to the yw pA-seq data, with the exception that we required that the editing site occur in both samples (2 of 2).

Editing levels of our identified Nascent sites were also calculated in 6 paired end 36bp sequenced mRNA samples, as well as 6 single end 72bp sequenced mRNA samples of the fly strain Canton-s (Cs). Editing levels were then calculated for the sites found in the nascent analysis.

Determination of Editing Level

For reproducibility of the nascent level, we pooled the editing counts for each replicate set of 6 time points together and calculated the percent editing level. The final percent editing level was determined by pooling the editing counts for all 12 samples and dividing by the total pooled counts of the 12 samples. Editing was similarly calculated for the yw pA-seq data and the small scale Nascent-seq data by pooling both replicate samples. The editing level for the Cs pA-seq data was calculated by pooling all 12 samples. A 2 tailed paired t-test was used to test the significance of the observed editing levels between the nascent and yw mRNA data (p = 1e-21).

Inferred Half-life

All genes were ranked by the pooled average of nascent read coverage per base pair and the pooled average of the yw mRNA read coverage per base pair. A ranking of 1 indicates the gene contains the highest number of reads per base pair. We observed that edited genes were among the most highly expressed in the Nascent-seq data, even when controlling for transcript length (Fig. S2C). The log base 2 ratio of the nascent Rank/mRNA Rank was calculated. We looked at a few functional categories of genes known to have long relative half-lives (Wang et al., 2002) and observed a good correlation with inferred half-lives (Fig. S2D). We observed a significant difference between edited and all genes (2 tailed t-test with unequal variances, p = 1e-81). Larger genes also had longer inferred half-lives by this log ratio metric (data not shown), therefore genes were grouped into 10 bins of 37 genes leaving out the 14 longest genes, and 11 ambiguous genes where the editing site falls into two genes on the same strand. Unique genes of similar or larger size were selected into respective background distribution bins (Fig. S2A). Only background genes with a minimum nascent average of 4 reads per base pair were considered. Because the variances of the log ratio metric were different between groups (Levene’s test of Error Variances, p <= 1e-4), we did not use an ANOVA test. Alternatively, using t-tests with unequal variance within the individual bins, we observed a significant difference between edited and background genes in 9 of 10 bins. (p<0.005, 2 tailed t-test with unequal variances, Bonferroni corrected alpha; Fig. S2B)

Edited Gene Overlap

Edited genes were classified into Exon, Intron, or Exon and Intron. A significant overlap was observed (2 tailed Fisher’s exact test, p = 1e-10) using all genes with a minimum NUN signal average of 2reads per base pair resulting in 7703 genes total genes.

Annotation of Edited Sites

Sites found in an alternatively spliced exon or within a UTR were assigned preferentially to exons and not introns. UTRs and splice sites were generated from annotation downloaded from the UCSC Genes and Gene Prediction group, flybaseGene Track/table Apr. 2006 assembly.

modENCODE, Hoopengardner, Jepson Data Preparation

Datasets were downloaded and filtered by using our genomic DNA criterion; removing sites which contained one or more Gs in the gDNA seq, or had lower than 10X coverage. We obtained 665 and 75 sites respectively.

Determining False Positive Rate

The exon and intron editing sites identified in the 12 Nascent-seq replicates were scanned in the small scale ADAR0, FM7a, yw Nascent-seq data. We required editing to occur in both replicates of the FM7a and yw data, and sites with zero sequence coverage in any sample were not considered. This resulted in 609 total editing sites: 374 exon sites and 235 intron sites. Editing levels from each of the 12 replicates of the main yw Nascent-seq data were used to determine the 5th percentile. Both replicates for each small scale (ADAR0, FM7a, yw) Nascent-seq dataset were pooled to determine the final editing level. If the editing level was greater than the 5th percentile it was considered a false positive. We then calculated the false positive rate.

Splicing Metrics

Intron retention, 5′ss and 3′ss ratio were calculated as described (Khodor et al., 2011). Briefly intron retention was calculated by dividing the average base pair coverage within the intron by the average base pair coverage within the exon. The 5′SS ratio is calculated as the average base pair coverage 25bp into the intron from the 5′ss, divided by the average base pair coverage 25bp into the exon from the 5′ss. The 3′ss was similarly calculated. Exon and intron UCSC annotation were handled as described above. Kruskal-Wallis non parametric tests were used to test significance between the groups.

HIGHLIGHTS

  • Most Drosophila RNA editing is cotranscriptional
  • Most cotranscriptional A-to-I events require ADAR
  • Editing within introns is widespread
  • Intron editing occurs preferentially on poorly spliced introns

Supplementary Material

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04

ACKNOWLEDGEMENTS

J.R., J.S.M., M.R. conceived and designed the experiments; J.R. performed the experiments and bioinformatic analyses; J.R., J.S.M., M.R. analyzed the data; J.R., J.S.M., M.R. wrote the manuscript. The authors declare no competing financial interests. We thank Katharine Abruzzi for helpful comments. We thank the Robert Reenan for kindly providing the ADAR0 fly strain. We thank Peter Bronk, Chris Vecsey and Leslie Griffith for fly strains. This research was supported by the Howard Hughes Medical Institute. J.R. was supported in part by a grant from the National Science Foundation (Integrative Graduate Education and Research Traineeship; DGE 0549390) and from the NIH (Genetics Training Grant; 5T32GM007122).

Footnotes

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ACCESSION NUMBERS

All raw sequencing data are available for download from NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE37232.

Additional methods are provided in Supplemental Information.

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