Fig. 1: DICER cleavage accuracy is dictated by 5′-nucleotide identity.
From: DICER cleavage fidelity is governed by 5′-end binding pockets
a,Schematic of pre-mir-517a substrates, illustrating terminal pairs (for example, G–U and C–U) at the 5′- and 3′-ends. Green and red arrowheads indicate DC21 and DC22 cleavage sites, located 21 and 22 nt from the 5′-end, producing fragments F1, F2 and F3. b, In vitro dicing assays for pre-mir-517a and variants using 2 pmol RNA and 1 pmol DICER. F1 and F3 fragments are 22 nt (DC22) or 21 nt (DC21), whereas F2 is undetectable on gels. c, Cleavage accuracy of DICER at DC21 and DC22 for pre-mir-517a variants, based on four independent experiments. Statistical significance was determined using a two-tailed, two-sample t-test (**P < 0.01, ****P < 0.0001). d, Schematic of massively parallel dicing assays using pre-mir-324 groups with fixed 5′-nt (A, G, U or C) and randomized 3′-overhang. Cleaved products (F3) were cloned, sequenced and analysed. e, Cleavage patterns from pre-mir-324 groups with distinct 5′-end nucleotides. f, Cleavage sites (DC20–DC23) identified from sequencing results. n = 256 variants. g, Cleavage accuracy at DC21 and DC22 for pre-mir-324 groups, calculated from sequencing data. n = 64 for each comparing group. h, Diagrams of pre-mir-629, pre-mir-208a and variants, showing terminal pairs. Green and red arrowheads indicate DC21 and DC22 cleavage sites. i, In vitro dicing assays for pre-mir-629, pre-mir-208a and variants using 2 pmol RNA and 1 pmol DICER. F1 and F3 fragments are 22 nt (DC22) or 21 nt (DC21); F2 is undetectable. Green arrowheads, DC21; red arrowheads, DC22. j, Cleavage accuracy of DICER at DC21 and DC22 for pre-mir-629 and pre-mir-208a variants, based on three independent experiments. Statistics as in c. k, Mechanistic model illustrating how 5′-nt affects DICER cleavage. Guanine (G) and adenine (A) favour DC21 cleavage, whereas uracil (U) and cytosine (C)—especially U—favour DC22 cleavage.
