Unique structural features of miRNAs

In the last two decades the small non-coding RNA field has significantly expanded beyond snRNAs & snoRNAs (1) to include piRNAs (2), siRNAs (3), novel small RNAs derived from known non-coding RNAs including tRNAs (4) and rRNAs (5), and, of course, microRNAs (miRNAs). Each of these types of small RNAs are characterized by a distinctive suite of characteristics and a unique evolutionary history. MiRNAs can be distinguished from other small genomically encoded RNA families by the following criteria (6 & Figure):

  1. Two 20-26nt long reads are expressed from each of the two arms derived from a hairpin precursor.

  2. Because the ends of canonical miRNA reads are generated enzymatically, the 5’ ends of the reads are homogeneous (>90%).

  3. The hairpin precursor shows imperfect complementarity, and base pairs in at least 16 of the ~ 22 nucleotides.

  4. The 5p and 3p reads are offset by 2 nucleotides on both ends due to the sequential processing of the miRNA 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 (7,8).

  5. 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 (6), even though most taxa like vertebrates with single Dicer genes never show loop lengths greater than ~40 nucleotides.

  6. There are other features of miRNAs, in particular structural and evolutionary signatures, that allow them to be further distinguished from other small RNAs:

  7. The mature miRNA 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) (6,9,10).

  8. 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) (6, 11).

  9. Processing motifs are often (but not always) present in the primary miRNA 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 (12,13).

Recognition and utilization of clear and, for the most part mechanistically well understood, criteria will allow the delineation of bona fide miRNAs from the myriad small RNAs generated in eukaryotic cells, allowing for deeper and robust insights into their function, possible mis-regulation, and evolution.


1. Matera AG, Terns RM, Terns MP. Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nature reviews Molecular cell biology 2007;8:209-20

2. Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP, et al. Characterization of the piRNA complex from rat testes. Science 2006;313:363-7

3. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999;286:950-2

4. Goodarzi H, Liu X, Nguyen HC, Zhang S, Fish L, Tavazoie SF. Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015;161:790-802

5. Chak LL, Mohammed J, Lai EC, Tucker-Kellogg G, Okamura K. A deeply conserved, noncanonical miRNA hosted by ribosomal DNA. Rna 2015;21:375-84

6. Fromm B, Billipp T, Peck LE, Johansen M, Tarver JE, King BL, et al. A Uniform System for the Annotation of Human microRNA Genes and the Evolution of the Human microRNAome. Annual review of genetics 2015;49:null

7. Kim B, Ha M, Loeff L, Chang H, Simanshu DK, Li S, et al. TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms. The EMBO journal 2015;34:1801-15

8. Kim YK, Kim B, Kim VN. Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis. Proceedings of the National Academy of Sciences of the United States of America 2016;113:E1881-9

9. Suzuki HI, Katsura A, Yasuda T, Ueno T, Mano H, Sugimoto K, et al. Small-RNA asymmetry is directly driven by mammalian Argonautes. Nature structural & molecular biology 2015;22:512-21

10. Schirle NT, Sheu-Gruttadauria J, MacRae IJ. Structural basis for microRNA targeting. Science 2014;346:608-13

11. Wheeler BM, Heimberg AM, Moy VN, Sperling EA, Holstein TW, Heber S, et al. The deep evolution of metazoan microRNAs. Evol Dev 2009;11:50-68

12. Nguyen TA, Jo MH, Choi YG, Park J, Kwon SC, Hohng S, et al. Functional Anatomy of the Human Microprocessor. Cell 2015;161:1374-87

13. Auyeung VC, Ulitsky I, McGeary SE, Bartel DP. Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing. Cell 2013;152:844-58