The fate of miRNA* strand through evolutionary analysis: implication for degradation as merely carrier strand or potential regulatory molecule?

Abstract

Background: During typical microRNA (miRNA) biogenesis, one strand of a approximately 22 nt RNA duplex is preferentially selected for entry into a silencing complex, whereas the other strand, known as the passenger strand or miRNA* strand, is degraded. Recently, some miRNA* sequences were reported as guide miRNAs with abundant expression. Here, we intended to discover evolutionary implication of the fate of miRNA* strand by analyzing miRNA/miRNA* sequences across vertebrates.

Principal findings: Mature miRNAs based on gene families were well conserved especially for their seed sequences across vertebrates, while their passenger strands always showed various divergence patterns. The divergence mainly resulted from divergence of different animal species, homologous miRNA genes and multicopy miRNA hairpin precursors. Some miRNA* sequences were phylogenetically conserved in seed and anchor sequences similar to mature miRNAs, while others revealed high levels of nucleotide divergence despite some of their partners were highly conserved. Most of those miRNA precursors that could generate abundant miRNAs from both strands always were well conserved in sequences of miR-#-5p and miR-#-3p, especially for their seed sequences.

Conclusions: The final fate of miRNA* strand, either degraded as merely carrier strand or expressed abundantly as potential functional guide miRNA, may be destined across evolution. Well-conserved miRNA* strands, particularly conservation in seed sequences, maybe afford potential opportunities for contributing to regulation network. The study will broaden our understanding of potential functional miRNA* species.

Figure 1. The selection of mature miRNA strand and the fate of its passenger strand.

(A) The mature miRNA sequence is miR-#-5p, and its passenger sequence is miRNA* (star) which is degraded. (B) The mature miRNA sequence is miR-#-3p, and its passenger sequence is miRNA*. (C) Both the miR-#-5p and miR-#-3p are mature miRNA sequences that guide RISC to silence target mRNAs through mRNA cleavage, translational repression or deadenylation.

Figure 2. Clustal multiple sequence alignment and mutational profile of miRNA and miRNA* sequences across vertebrates.

(A) Multiple sequence alignment of mir-124 family and mutational profile of miRNA and miRNA* sequences. Repeated sequences were discarded. Conserved: conserved positions in sequence alignment. The bottom figures were the percentage of nucleotide substitutions from positions 1 to ∼22 in miRNA and miRNA* sequences. Both miR-124 and miR-124* sequences were well conserved, especially miR-124. (B) Mutational profile of miRNA/miRNA* in miRNA gene families. Both miRNA and miRNA* sequences showed different evolutionary patterns. miRNA* sequences always were involved more nucleotide substitutions than miRNAs, even in positions 2–8. (C) Mutational profile of miR-#-5p/miR-#-3p in miRNA gene families. These miRNAs (miR-#-5p and miR-#-3p) were reported as abundant miRNAs according to miRBase database (version 14.0). Although miR-#-5p had different divergence pattern with miR-#-3p, both of them always were well conserved especially for the seed sequences. In mir-199 family, nucleotide divergences of miR-199-5p mainly resulted from gga-mir-199b and bta-mir-199c.

Figure 3. Clustal multiple sequence alignment of let-7 family and mutational profile of miRNA and miRNA* sequences.

(A) Hsa-let-7 showed high conservation in positions 2-8 (the seed sequence) and relatively conservation in positions 13–16, while miRNA* sequence showed frequent substitutions and insertions/deletions. (B) Let-7 sequences across vertebrates showed high conservation in positions 2–8 (the seed sequence), while let-7* sequences showed frequent substitutions.

Figure 4. Phylogenetic network of hsa-let-7 family using neighbor-net method.

Based on miRNA precursor sequences of let-7 family in Homo sapiens, 12 miRNA genes were split into several clusters. Multicopy miRNA precursors for a single miRNA, for example, hsa-let-7a-1, hsa-let-7a-2 and hsa-let-7a-3, were reconstructed in different clusters.

Figure 5. Nucleotide divergences of miRNA and miRNA* across several typical vertebrates.

In order to avoid influence of various distribution spectrums of miRNAs, we selected several typical vertebrates to analyze miRNA evolutionary trends: dre (Danio rerio, Pisces), hsa (Homo sapiens, Mammalia), gga (Gallus gallus, Aves) and xtr (Xenopus tropicalis, Amphibia). Red conserved sites indicated mature miRNAs (including less abundant but functional miRNA* strands), pink conserved sites indicated miRNA* sequences that were not reported as functional guide miRNAs. Some flank sequences of miRNA sequences were deleted. All the mature miRNAs were highly conserved in these animals, while their miRNA* strands showed different divergence trends. (A) miRNA* sequences were less conserved due to divergence of different animals and multicopy miRNA precursors. (B) miRNA* sequences were well conserved similar to mature miRNAs though they were not reported as abundant functional miRNAs. (C) miR-#-5p and miR-#-3p were well conserved across different animals, and both of them were reported as abundant functional guide miRNAs.