Unique structural features of microRNAs
In the last three decades, the small non-coding RNA field has significantly expanded beyond snRNAs & snoRNAs (1) to include piRNAs (2–4), siRNAs (5, 6), novel small RNAs derived from known non-coding RNAs including tRNAs (7) and, of course, microRNAs (8–11). Each of these types of small RNAs are characterized by a distinctive suite of characteristics and a unique evolutionary history. microRNAs can be distinguished from other small genomically encoded RNA families by the following criteria (12–14) & Figure):
Two 20-26nt long reads are expressed from each of the two arms derived from a hairpin precursor.
Because the ends of canonical microRNA reads are generated enzymatically, the 5’ ends of the reads are homogeneous (>90%).
The hairpin precursor shows imperfect complementarity, and base pairs in at least 16 of the ~ 22 nucleotides.
The 5p and 3p reads are offset by 2 nucleotides on both ends due to the sequential processing of the microRNA transcript by Drosha and Dicer to generate the mature ~22 nucleotide read(s). In some cases, the Drosha offset is only offset by 1 templated nucleotide, but in these cases the 3’ end of the 3p arm is monouridilyated (15, 16).
The length of the loop is at least 8 nucleotides long; there is no apparent maximum in loop length, even in organisms possessing only a single Dicer gene, contra (13), even though most taxa like vertebrates with single Dicer genes never show loop lengths greater than ~40 nucleotides.
The mature microRNA sequence usually starts with A or U, and is often mismatched with the complementary arm, which seems to facilitate arm selection by Argonaute (at least in mammals) (17,18,19).
Nucleotide positions 2 through 8 of the mature sequence (the "seed") are strongly conserved through evolution, as are positions 13-16 (the 3' complementary region) (13,20,21).
Processing motifs are often (but not always) present in the primary microRNA transcript including a UG motif 14 nucleotides upstream of the 5p arm, a UGU motif at the 3' end of the 5p arm, and a CNNC motif 17 nucleotides downstream of the 3p arm (22,23).
There are other features of microRNAs, in particular structural and evolutionary signatures, that allow them to be further distinguished from other small RNAs:
Recognition and utilization of clear and, for the most part mechanistically well understood, criteria will allow the delineation of bona fide microRNAs from the myriad small RNAs generated in eukaryotic cells, allowing for deeper and robust insights into their function, possible mis-regulation, and evolution.
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