Limited RNA editing in exons of mouse liver and adipose

Abstract

Several studies have investigated RNA-DNA differences (RDD), presumably due to RNA editing, with conflicting results. We report a rigorous analysis of RDD in exonic regions in mice, taking into account critical biases in RNA-Seq analysis. Using deep-sequenced F1 reciprocal inbred mice, we mapped 40 million RNA-Seq reads per liver sample and 180 million reads per adipose sample. We found 7300 apparent hepatic RDDs using a multiple-site mapping procedure, compared with 293 RDD found using a unique-site mapping procedure. After filtering for repeat sequence, splice junction proximity, undirectional strand, and extremity read bias, 63 RDD remained. In adipose tissue unique-site mapping identified 1667 RDD, and after applying the same four filters, 188 RDDs remained. In both tissues, the filtering procedure increased the proportion of canonical (A-to-I and C-to-U) editing events. The genomic DNA of 12 RDD sites among the potential 63 hepatic RDD was tested by Sanger sequencing, three of which proved to be due to unreferenced SNPs. We validated seven liver RDD with Sequenom technology, including two noncanonical, Gm5424 C-to-I(G) and Pisd I(G)-to-A RDD. Differences in diet, sex, or genetic background had very modest effects on RDD occurrence. Only a small number of apparent RDD sites overlapped between liver and adipose, indicating a high degree of tissue specificity. Our findings underscore the importance of properly filtering for bias in RNA-Seq investigations, including the necessity of confirming the DNA sequence to eliminate unreferenced SNPs. Based on our results, we conclude that RNA editing is likely limited to hundreds of events in exonic RNA in liver and adipose.

Figure 1

RDD in mouse liver and adipose identified by RNA-Seq. (A) RDD numbers resulting from different filters. The results found for the same filters by Kleinman and Majewski (2012) are in italics. (B) Percentages of RDD reads found in comparison with reads from the EST database.

Figure 2

Contribution of sequencing strand bias to RDD. Histogram with the distribution of RDD (y-axis) according to the proportion of reads belonging to the forward sequencing strand (x-axis) in liver (sample M.CH.BxD) and adipose (sample F.BxD). Open bars and the dashed line correspond to the RDD sites. Shaded bars and the solid line correspond to the dbSNP sites that are polymorphic in the sample and used as a control data set. The false positives are calculated using the tails of the distributions where the RDD and SNP distributions intersect. The same shape of distribution was observed for all samples of the same tissue.

Figure 3

Contribution of end of read sequencing to RDD. Histogram with the distribution of RDD (y-axis) according to the proportion of reads where the alternative base is found in the first or last base or in the 5 first or 5 last bases of the sequencing read (x-axis). (A) Liver (sample M.CH.BxD) and (B) adipose (sample F.BxD). Open bars and the dashed line correspond to the RDD sites. Shaded bars and the solid line correspond to the dbSNP sites that are polymorphic in the sample and used as a control data set. The false positives are calculated using the tails of the distributions where the RDD and SNP distributions intersect. The same shape of distribution was observed for all samples of the same tissue.

Figure 4

RDD categories in liver and adipose tissue. (A) RDD categories observed with liver RNA-Seq data after the multiple and unique mapping procedures and filtering for sequencing bias. (B) RDD categories observed with adipose RNA-Seq data after the unique mapping procedure and filtering for sequencing bias.

Figure 5

Validation by Sequenom technology of four RDD observed by RNA-Seq in liver. (A) Results obtained by Sequenom technology and expressed as percentage of total mRNA sequences containing the DNA base (open) vs. the edited base (solid) (total, 100%). (B) Results obtained by RNA-Seq technology and expressed as number of reads mapping to the RDD position.