Identification and characterization of novel amphioxus microRNAs by Solexa sequencing

Abstract

Background: microRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. While the number of known human and murine miRNAs is continuously increasing, information regarding miRNAs from other species such as amphioxus remains limited.

Results: We combined Solexa sequencing with computational techniques to identify novel miRNAs in the amphioxus species B. belcheri (Gray). This approach allowed us to identify 113 amphioxus miRNA genes. Among them, 55 were conserved across species and encoded 45 non-redundant mature miRNAs, whereas 58 were amphioxus-specific and encoded 53 mature miRNAs. Validation of our results with microarray and stem-loop quantitative RT-PCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. Analyzing the evolutionary history of amphioxus miRNAs, we found that amphioxus possesses many miRNAs unique to chordates and vertebrates, and these may thus represent key steps in the evolutionary progression from cephalochordates to vertebrates. We also found that amphioxus is more similar to vertebrates than are tunicates with respect to their miRNA phylogenetic histories.

Conclusions: Taken together, our results indicate that Solexa sequencing allows the successful discovery of novel miRNAs from amphioxus with high accuracy and efficiency. More importantly, our study provides an opportunity to decipher how the elaboration of the miRNA repertoire that occurred during chordate evolution contributed to the evolution of the vertebrate body plan.

Figure 1

Step-by-step schematic description of the strategy for amphioxus miRNA discovery and validation. nt = nucleotide; snRNA = small nuclear RNA; small nucleolar RNA.

Figure 2

Characterization of amphioxus miRNAs. (a, b) Comparison of the number (a) and absolute sequencing frequency (b) of conserved miRNAs with those of amphioxus-specific miRNAs. (c) The composition of amphioxus miRNAs of various lengths (in nucleotides (nt)). (d) The size distribution of small amphioxus RNAs and miRNAs of various lengths sequenced by Solexa.

Figure 3

Confirmation of the accuracy of Solexa sequencing with qRT-PCR and microarray analysis. (a) The expression levels of the indicated miRNAs in amphioxus evaluated by semi-quantitative RT-PCR with 30 cycles. (b-d) Nine miRNAs (bbe-miR-1, bbe-miR-10a, bbe-miR-29b, bbe-miR-92a, bbe-miR-125, bbe-miR-184, bbe-miR-210, bbe-miR-216, and bbe-miR-217) were selected and their expression levels were measured by Solexa sequencing, stem-loop qRT-PCR and microarray analysis. The data obtained from each of these methods were then compared with the data obtained from each of the others and drawn as a Pearson correlation scatter plot.

Figure 4

The phylogenetic histories of amphioxus miRNAs. (a) miRNA gene clusters in amphioxus. At a 3,000-nucleotide distance threshold, the amphioxus genome contains 17 compact clusters with 39 miRNAs. The precursor structure is indicated as a box, and the location of the miRNA within the precursor is shown in black. Some of these clusters in amphioxus are also conserved in zebrafish and humans. (b) The evolutionary histories of miRNAs and their relationship to the milestones of macroevolution. We integrated amphioxus miRNAs into the currently known miRNAs (miRBase release 12.0) and performed a comprehensive screening of their phylogenetic histories across animals. Each miRNA was classified into one of four groups: miRNAs conserved throughout bilaterian animals; homologs of invertebrate miRNAs; miRNAs present in both chordates and vertebrates; and homologs of vertebrate miRNAs. Note that our approach ignored species-specific miRNAs, since these miRNAs do not offer any information about miRNA evolution. (c) Comparison of the miRNA repertoires of amphioxus and tunicates. By using zebrafish as a reference, we compared the miRNA repertoires of nematodes, fruit flies, tunicates, and amphioxus. miRNAs with a zebrafish homolog were recorded as +1; miRNAs not found in zebrafish were recorded as -1.