Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications

Abstract

Recent advances in DNA-sequencing technology have made it possible to obtain large datasets of small RNA sequences. Here we demonstrate that not all non-perfectly matched small RNA sequences are simple technological sequencing errors, but many hold valuable biological information. Analysis of three small RNA datasets originating from Oryza sativa and Arabidopsis thaliana small RNA-sequencing projects demonstrates that many single nucleotide substitution errors overlap when aligning homologous non-identical small RNA sequences. Investigating the sites and identities of substitution errors reveal that many potentially originate as a result of post-transcriptional modifications or RNA editing. Modifications include N1-methyl modified purine nucleotides in tRNA, potential deamination or base substitutions in micro RNAs, 3′ micro RNA uridine extensions and 5′ micro RNA deletions. Additionally, further analysis of large sequencing datasets reveal that the combined effects of 5′ deletions and 3′ uridine extensions can alter the specificity by which micro RNAs associate with different Argonaute proteins. Hence, we demonstrate that not all sequencing errors in small RNA datasets are technical artifacts, but that these actually often reveal valuable biological insights to the sites of post-transcriptional RNA modifications.

The use of high-throughput sequencing methods for plant microRNA research

“…[67][68][69][70] However, recent research progresses point to the potential involvement of RNA editing in the modifications of plant nuclear transcripts. 59,71 More interestingly, in both animals and plants, RNA modifications were discovered on the miRNA precursors and the mature miRNAs. 59,[71][72][73] Considering the fact that the featured "stem-loop" structures are critical for processing of the miRNA precursors, RNA editing might alter the conformation and the stability of the secondary structures of the miRNA precursors, thus affecting their processing efficiency and the miRNA abundances.…”

“…59,71 More interestingly, in both animals and plants, RNA modifications were discovered on the miRNA precursors and the mature miRNAs. 59,[71][72][73] Considering the fact that the featured "stem-loop" structures are critical for processing of the miRNA precursors, RNA editing might alter the conformation and the stability of the secondary structures of the miRNA precursors, thus affecting their processing efficiency and the miRNA abundances. Besides, RNA editing occurred on mature miRNAs or their target transcripts could have a great impact on the stability of miRNA-target interactions (Fig.…”

“…However, these discarded "error" reads were utilized by Ebhardt et al 71 for study on the post-transcriptional modifications of tRNAs and mature miRNAs in plants. 71 They hypothesized that some of the "error" reads might have their biological origins.…”

“…71 They hypothesized that some of the "error" reads might have their biological origins. The discrepancies between the "error" reads and the genomic sequences could originate from post-transcriptional modifications.…”

“…In contrast to the random occurrences of technical sequencing errors, the site-specific modifications or editing events should be repeatedly observed at single sites with much higher frequencies. 71 In other words, a reliable editing site should be supported by a certain number of sRNA-seq reads with a fixed mismatch site compared to the genomic site (Fig. 1).…”