microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5′ start but common at the 3′ end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5′ isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5′ isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5′ most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.

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SummaryEmerging findings indicate that cells can produce both micro (mi)RNAs and their messenger (m)RNA targets in multiple processing variants in a tissue-and developmental stage-selective manner. Specifically, we find that cells accumulate a greater range of functional miRNAs than hitherto expected, whereas mRNAs with alternative 3 0 untranslated regions that include varying numbers of miRNA target sites are also seen to be common. This has important implications for both our understanding of miRNA function in a given biological context and the design of successful strategies for experimental or therapeutic intervention. In this review, we relate these new phenomena to miRNAs in the heart, where they are known to play critical roles during normal function as well as in cardiac disease. IUBMBIUBMB Life, 64(11): 872-878, 2012 Keywords cardiac disease; miRNA biogenesis; miRNA-target interaction; 3 0 untranslated region; mRNA 3 0 end cleavage; polyadenylation.

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