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

Abstract

Summary 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.

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

“…S3B,C). This is consistent with earlier observations from the Kim group and recent findings by the Bartel group (Han et al 2006;Fang and Bartel 2015). Unexpectedly, we also observed two bulge-depleted regions in miRNA hairpins, located at positions ∼5-9 nt and ∼16-21 nt relative to apical loop, or ∼16-21 nt and ∼28-32 nt relative to the base of the hairpin, observable for both arms of the stem (Fig.…”

“…For example, several studies have proposed that the Microprocessor complex efficiently processes hairpins with stem lengths of ∼33 nucleotides (nt) (Han et al 2006;Nguyen et al 2015). Another recent study has used a cell-free screen on more than 210,000 random mutations in three pri-miRNA hairpins and found that the most favored substrates of the Microprocessor complex are hairpins of ∼35 ± 1 nt in stem length (Fang and Bartel 2015). Apical loop sizes of 3-23 nt have been found to be compatible with processing (Zeng and Cullen 2003), with loop size of ≥10 nt proposed to allow efficient processing (Ma et al 2013).…”

“…Apical loop sizes of 3-23 nt have been found to be compatible with processing (Zeng and Cullen 2003), with loop size of ≥10 nt proposed to allow efficient processing (Ma et al 2013). In addition, a bulge was often found near the DROSHA processing site several bases from the base of the hairpin (Han et al 2006) and, in the context of a GHG motif, can enhance the processing of specific miRNAs (Fang and Bartel 2015).…”

“…Other than the CNNC motif, the UG motif at the basal stem junction and UGU/GUG/ UGUG motif in the apical loop can be found in a minor subset of miRNAs (Auyeung et al 2013;Nguyen et al 2015). The relationship between structural features and primary sequence motifs has been examined in a recent study, which proposes that a combination of primary sequence motifs (CNNC, basal UG, and apical UGU/GUG/UGUG) enhances the processing of hairpins with nonoptimal lengths (Fang and Bartel 2015). Aside from structural and sequence features in pri-miRNAs, a number of protein factors have been shown to regulate processing of specific miRNAs or miRNA subsets (Denli et al 2004;Gregory et al 2004;Yang et al 2006;Guil and Caceres 2007;Davis et al 2008Davis et al , 2010Newman et al 2008;Viswanathan et al 2008;Paroo et al 2009;Trabucchi et al 2009;Nam et al 2011;Piskounova et al 2011;Tang et al 2011;Kawahara and Mieda-Sato 2012;Wada et al 2012;Di Carlo et al 2013;Cheng et al 2014).…”

Drosha and Dicer: Slicers cut from the same cloth

“…Based on previous biochemical data, it has been proposed that Microprocessor cleaves at a distance of ~11-bp from the basal junction and at a distance of ~22-bp from the apical junction. Recent studies have identified four primary sequence determinants located in the pri-miRNA scaffold (UGUG element in apical loop, GHG (H = A, U or C) elements in the stem, and UG and CNNC elements in the basal ssRNA overhangs ( Figure 1B)) that contribute to cleavage specificity and efficiency [5], providing additional constraints on Microprocessor recognition.…”