Substrate promiscuity of Dicer toward precursors of the let-7 family and their 3′-end modifications

The human let-7 miRNA family consists of thirteen members that play critical roles in many biological processes, including development timing and tumor suppression, and their levels are disrupted in several diseases. Dicer is the endoribonuclease responsible for processing the precursor miRNA (pre-miRNA) to yield the mature miRNA, and thereby plays a crucial role in controlling the cellular levels of let-7 miRNAs. It is well established that the sequence and structural features of pre-miRNA hairpins such as the 5′-phosphate, the apical loop, and the 2-nt 3′-overhang are important for the processing activity of Dicer. Exceptionally, nine precursors of the let-7 family (pre-let-7) contain a 1-nt 3′-overhang and get mono-uridylated in vivo, presumably to allow efficient processing by Dicer. Pre-let-7 are also oligo-uridylated in vivo to promote their degradation and likely prevent their efficient processing by Dicer. In this study, we systematically investigated the impact of sequence and structural features of all human let-7 pre-miRNAs, including their 3′-end modifications, on Dicer binding and processing. Through the combination of SHAPE structural probing, in vitro binding and kinetic studies using purified human Dicer, we show that despite structural discrepancies among pre-let-7 RNAs, Dicer exhibits remarkable promiscuity in binding and cleaving these substrates. Moreover, the 1- or 2-nt 3′-overhang, 3′-mono-uridylation, and 3′-oligo-uridylation of pre-let-7 substrates appear to have little effect on Dicer binding and cleavage rates. Thus, this study extends current knowledge regarding the broad substrate specificity of Dicer and provides novel insight regarding the effect of 3′-modifications on binding and cleavage by Dicer. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-05090-2.


Fig. S1
(cyan, Dicer-RNA complex with a full RNA stem) 5ZAM (green-yellow, Dicer-RNA complex with a partial RNA stem) are shown [5].c Alignment of the PPC domain from the cryo-EM structures of human Dicer free and in complex with pre-let-7a1 GYM .PDB entries 7XW3 (green, Dicer only) and 7XW2 (pink, Dicer-RNA complex in a dicing state) are shown [6].d Alignment of PPC domain from the cryo-EM structures of mouse Dicer in complex with pre-miR-15a.PDB entries 7YZ4 (orange, Dicer only), 7YYM (raspberry, pre-dicing state), and 7YYN (yellow, Dicing state) are shown [7].The platform-PAZ-connector (PPC) were aligned for all structures and the regions of interaction between the PAZ and RNA substrates are shown.
Fig. S1 Secondary structures of the lowest-energy conformers for selected pre-let-7 RNAs determined by SHAPE.The structures shown represent the lowest energy conformation(s), using a threshold of maximum energy difference with the lowest energy structure of 50% (Emax = 50%).The energies of the structure provided by the RNAstructure software are given in kcal/mol.The normalized 1M7 reactivity of each nucleotide is color-coded as per the SHAPE reactivity key.Dicer cleavage sites are indicated with an arrowhead.

Fig. S3
Fig. S3 Quality assurance of purified RNA substrates and enzyme for this study.a Schematic representation of a typical T7 RNA transcript containing the pre-let-7 substrate flanked by the hammerhead (HH) and Hepatitis delta virus (HDV) ribozymes.The HH ribozyme is engineeredfor each pre-let-7 miRNA and added to the 5' end, whereas the HDV ribozyme is attached to the 3' end[3].b Self-cleavage of the T7 RNA transcript into smaller fragments during in vitro transcription.The smaller RNA fragments (pre-let-7 RNA, HH ribozyme, and HDV ribozyme) are separated by electrophoresis using a 10% denaturing polyacrylamide gel, and the pre-let-7 RNA is purified, as described in the Material and Methods section.The 84-nt RNA control on the gel is a purified version of the HDV ribozyme.c Purified pre-let-7 substrates with homogenous 5' and 3'ends.d SDS-PAGE characterization (7.5% gel) of purified Dicer before and after incubation at 37°C.

Fig. S4
Fig. S4 Characterization of miRNA-5p products for Dicer cleavage of pre-let-7 substrates.Cleavage assays for 12 pre-let-7 RNAs were performed with (+) and without Dicer (-) under single-turnover conditions with 0.1 nM substrate and 5 nM Dicer for 15 min at 37 ºC.The cleavage reactions along with the T1 and OH -ladders generated from pre-let-7f-1 were analyzed by denaturing PAGE (right panel).The green arrowhead points to the standard Dicer cleavage site (22-nt).To help identify RNase T1 cleavage products on the denaturing gel, the primary and secondary structures of pre-let-7f-1 are shown (left panel) with the G residues numbered according to their position in the sequence.

Fig. S5
Fig. S5 Effect of TRBP on pre-let-7 cleavage by Dicer.a Cleavage assays for pre-let-7a-1 and prelet-7a-1_U were performed under single turnover conditions (0.1 nM substrate and 5 nM Dicer) in the absence and/or presence of TRBP (5 nM), and the kobs values were derived with standard deviation (shown by the error bars) from the exponential fits of the pseudo-first order cleavage data from at least three independent experiments.b Cleavage assays for pre-let-7a-1 and pre-let-7a-1_U under multiple-turnover conditions (25 nM substrate and 0.5 nM Dicer) in the absence and/or presence of TRBP (10 nM).The standard deviations (shown by the error bars) were obtained from at least three independent experiments.In (a) and (b), the following symbols are used for cleavage under different conditions: circles for pre-let-7a-1 without TRBP; squares for pre-let-7a-1 with TRBP; and triangles for pre-let-7a-1_U with TRBP.

Fig. S6
Fig. S6 Characterization of miRNA-5p products for Dicer cleavage of uridylated and adenylated pre-let-7a-1 substrates.Cleavage assays for pre-let-7a-1, uridylated pre-let-7a-1, and adenylated pre-let-7a-1 were performed with (+) and without Dicer (-) under single-turnover conditions (0.1 nM substrate and 5 nM Dicer) for 15 min at 37ºC.The cleavage reactions along with the T1 and OH -ladders generated from pre-let-7a-2 were analyzed by denaturing PAGE (right panel).The green arrowhead points to the standard Dicer cleavage site (22-nt) and the red arrow indicates the alternative Dicer cleavage site (17 nt).To help identify RNase T1 cleavage products on the denaturing gel, the primary and secondary structures of pre-let-7a-2 are shown (left panel) with the G residues numbered according to their position in the sequence.

Fig. S7
Fig. S7 Time course for Dicer cleavage of pre-let-7a-1_6U under single-turnover conditions.aThe cleavage assay for pre-let-7a-1_6U was performed under single-turnover conditions using 0.1 nM RNA and 5 nM Dicer.The aliquots were analyzed on 15% denaturing PAGE.The two products, P1 and P2, are indicated with green and red arrowheads, respectively.b The percentage of product formation at several time-points were determined for both the P1 (green) and P2 (red) products individually and for the sum of products (P1 + P2: in grey).The exponential fit (not shown) of the single-turnover kinetic data for substrate cleavage (P1 + P2) yields the values of kobs (0.17 min -1 ) and maximum percentage of cleavage (96.2 %) with an R 2 value of 0.998.