The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes

• Published in: Molecular cell 2015-10, Vol.60 (1), p.131-145
• Main Authors: Fang, Wenwen, Bartel, David P.
• Format: Article
• Language: English
• Subjects: Computational Biology - methods; DEAD-box RNA Helicases - metabolism; genes; HEK293 Cells; Humans; microRNA; MicroRNAs - metabolism; Models, Molecular; Nucleic Acid Conformation; Ribonuclease III - metabolism; RNA Precursors - chemistry; RNA Precursors - metabolism; RNA Processing, Post-Transcriptional
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2015.08.015

MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base pairing throughout the remainder of the stem, and a narrow stem-length preference of 35 ± 1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence yet processed more efficiently than natural pri-miRNAs. [Display omitted] •High-throughput analyses of pri-miRNAs reveal determinants of efficient processing•Stem pairing and a length of 35 base pairs are key structural features•A mismatched motif and primary-sequence motifs compensate for structural defects•These defining features of pri-miRNAs enable reliable de novo design of miRNA genes MicroRNAs are processed from the stem-loop regions of primary microRNAs (pri-miRNAs). Using high-throughput analyses of pri-miRNA variants, Fang and Bartel identified features of pri-miRNA stems that promote processing. Analyses of these and previously identified features provided a unifying model defining pri-miRNAs, which enabled de novo design of functional miRNA genes.