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. 1999 May 25;96(11):6048-53.
doi: 10.1073/pnas.96.11.6048.

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

D P Morse  1 , B L Bass
Affiliations

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

D P Morse et al. Proc Natl Acad Sci U S A. .

Abstract

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

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Figures

Figure 1
Figure 1
A protocol for identifying endogenous ADAR substrates. (A) N, nucleotide 5′ of poly(A) tail. X, nucleotide(s) on the 3′ end of the downstream PCR primer (see Methods). Step 1: Poly(A)+ RNA is specifically cleaved 3′ of inosines as described (15). Step 2: A poly(A) tail is added 3′ to the inosine at the cleavage site to create a primer binding site for first-strand cDNA synthesis. Step 3: Because inosine pairs with cytidine, reverse transcription is performed with a T12C primer to enrich for cleaved molecules. The question mark indicates that the primer will extend on uncleaved RNA only if n = G. Step 4: Multiple aliquots of the cDNA are analyzed by using numerous combinations of primers (see Methods for primer design). Importantly, bands that derive from inosine-containing molecules depend on the addition of RNase T1 and are identified by comparison to samples not treated with ribonuclease. (B) Optimization of the method. A 386-nt synthetic RNA (0.5 fmol) containing a single inosine (15) was spiked into 5 μg of total yeast RNA and subjected to the protocol of A by using 0, 30, or 300 units of RNase T1. The complementarity between the upstream arbitrary PCR primer, and its priming site is shown above the gel; N represents a randomized position. For the downstream PCR primer, X=G because the nucleotide on the 5′ side of the inosine in the control RNA was a C. The arrow points to an RNase T1-dependent band whose sequence confirmed that it derived from the synthetic RNA cleaved precisely 3′ to its single inosine.
Figure 2
Figure 2
A differential display gel from the analysis of C. elegans poly(A)+ RNA. A region containing a T1-dependent band (boxed) is enlarged below. Each pair of lanes corresponds to a different primer pair, minus (Left) or plus (Right) RNase T1 (100 units). The dots on each side of the band are pinholes used to mark its position for excision and elution of the DNA. The T1-dependent band was called 9A because it was generated by using upstream arbitrary primer 9 and the GAGACCAGT12CA downstream primer; this nomenclature was used for all T1-dependent bands. M, DNA ladder with numbers in bp.
Figure 3
Figure 3
The predicted structure for each ADAR substrate is shown above its sequence, with observed editing sites shown in color. Ten cDNAs were sequenced for 1TC and 52G and six for all others. Adenosines edited in >70% of the cDNAs are red, those in 40–70% blue, and less than 40% green. For each cDNA, the percentage of total adenosines that appeared as guanosines was determined. 1TC (0–3%), 9A (2–7%), and 16G (0–4%) were selectively edited, whereas 52G (10–22%) and M05B5.5 (23–32%) were deaminated more promiscuously. ∗, RNase T1 cleavage sites detected by differential display; arrowhead (1TC), Tc1 insertion site; question mark (16G), unfinished sequence not yet confirmed by our independent sequencing. The most efficiently edited A in 1TC is underlined. Conclusions about editing site locations in M05B5.3, 9A, and 16G are based on ORFs predicted by genefinder and on our cDNA sequencing. cDNA sequences revealed that two introns predicted by genefinder are not removed by splicing. These are the predicted first intron of M05B5.3 and the predicted last intron of ZC239.6 (9A). Although the edited region in the pop-1 (1TC) message was previously reported to lie within an intron (23), our PCR and Northern blot analyses indicate the region is in the 3′ UTR. cDNA sequences corresponding to 7339–8109 of F55A4 showed 7339 is an SL2 trans-splicing site and 7681–7225 is an intron. A likely polyadenylation signal begins at 8178 that would generate a transcript of a size consistent with Northern analyses. The number of nucleotides sequenced beyond the ends of the indicated structures were as follows (5′, 3′): 1TC (167, 203), 9A (370, 91), and MO5B5.3 (, 74).
Figure 4
Figure 4
Limited primer extension assay. (A) Principle: 5′ end-labeled primers (arrows) are extended in the presence of three deoxynucleotides and one dideoxynucleotide (see Methods). With ddA, primers annealed to unedited molecules are extended to the editing site (∗), whereas primers annealed to edited molecules continue to the next A (sense strand). With ddG, edited molecules show a new stop compared with unedited sequences. Dashed arrows indicate primer extension. (B) An autoradiogram showing primer extension products from 9A cDNA or genomic DNA (gDNA). Additional bands in the cDNA lanes verified that the RNase T1 cleavage site (∗) and the next adenosine (∗∗) were edited. Quantification indicated 48% of the 9A RNA molecules were edited at the T1 cleavage site (∗). In similar experiments we quantified the T1 cleavage site of 16G (5%) and the most efficiently edited site in 1TC (25%; underlined, Fig. 3). Assays of the T1 cleavage site in 52G gDNA and cDNA verified the editing site and showed it was edited ≥25%. However, the 52G structure made cDNA synthesis so inefficient that trace amounts of contaminating gDNA were coamplified to become a significant fraction of the sample; thus an exact number could not be obtained.
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