The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1

Abstract

The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.

Fig. 1.

Recombinant DCL1 cleavage of dsRNA. (A) Domains of DCL1, HYL1, and SE. The 6× His, HA, and FLAG epitope tags are indicated. (B) Purified recombinant His-HA-DCL1-FLAG, His-HYL1, and His-SE proteins were fractionated on 4–12% SDS/PAGE and stained with Coomassie blue G-250. (C–F) DCL1 cleavage of dsRNA substrates. Recombinant DCL1 protein was incubated with 200 ng of a 94-bp dsRNA substrate with a 2-nt 3′ overhang (see Methods). The reaction products were phenol/chloroform purified and analyzed on 15% 8 M urea PAGE. (C) Time course of cleavage using 200 ng of DCL1. (D) dsRNA cleavage as a function of DCL1 protein concentration for 60 min. (E) The effect of NaCl concentration on dsRNA cleavage for 60 min with 200 ng of DCL1. (F) The effect of ATP and Mg²⁺ on DCL1 cleavage of dsRNA. EDTA (2 mM) was added to the reaction lacking the Mg²⁺ (last lane). The arrowheads indicates the dsRNA substrate, and the arrows indicate the 21-nt RNA products.

Fig. 2.

HYL1 and SE facilitate DCL1 cleavage of pre-miRNA. (A) Schematic representation of the miR167b pre-miRNA substrate. (B) DCL1 cleavage of the miR167b pre-miRNA substrate. Ten nanograms of the 5′ end-labeled 93-nt pre-miRNA were incubated with 15 ng of recombinant DCL1 for the indicated time. Recombinant HYL1 (150 ng) and recombinant SE (150 ng) were added to the reactions as indicated. Reactions without recombinant proteins (first lane) and with HYL1 (150 ng) and SE (150 ng), but without DCL1, were used as controls (last lane). (C) Quantification of the miR167b small cleavage products of the reactions shown in B. The activity was defined as the relative intensity of the band corresponding to the small RNA cleavage products (quantified by using ImageJ software). The ordinate shows the ratio of the intensity of the small RNA band observed with each combination of proteins to that observed with DCL1 alone at 1 min (lane 2). (D) The effect on DCL1 activity of varying amounts of HYL1 and SE singly and together. HYL1 and SE were each added singly or together at 40, 75, and 150 ng to a reaction containing 10 ng of the pre-miR167b substrate and 15 ng of DCL1. Control reactions (lanes 1–3) contained reaction buffer only or HYL1 (150 ng) or SE (150 ng) only. All of the reactions were incubated for 2 min at 37°C. (E) The 21-nt miRNA products were quantified as described in D and expressed as a ratio of the intensity of the band obtained with each combination of proteins to that observed with DCL1 alone (lane 4). The arrowheads indicates the pre-miR167b substrate, and the arrows indicates the cleavage products. D, DCL1; H, HYL1; S, SE.

Fig. 3.

In vitro processing of pri-miR167b. (A) Schematic representation of the pri-miR167b substrate. (B) RNA digestion products fractionated on an 8 M urea denaturing gel and stained with Sybr gold. The pri-miR167b substrate (125 ng) was incubated with 40 ng of recombinant DCL1 protein alone (lane 4) or with increasing amounts of either HYL1 (lanes 5–7, 30 ng, 60 ng, and 120 ng) or SE (lanes 8–10, 20 ng, 40 ng, and 80 ng) alone or both together (lanes 11–13, HYL1 at 30 ng, 60 ng, and 120 ng and SE at 20 ng, 40 ng, and 80 ng). Controls (lanes 1–3) contained reaction buffer, HYL1 only (120 ng), and SE only (80 ng), respectively. (C–D) Northern blots of RNA digestion products from the gel in B probed with the P³² end-labeled anti-miR167b (C) and anti-miR167b* (D) oligonucleotides, respectively. (E) Quantification of ≈21-nt RNA products shown in B by using ImageJ software. The ordinate shows the ratio of the intensity of the small RNA band observed with various combinations of proteins to that observed with DCL1 alone (lane 4). (F and G) Quantification of 21-nt bands detected by anti-miR167b (F) or anti-miR167b* (G) probes as described in E. Arrowheads, pri-miR167b substrates; asterisk, ≈21-nt RNA products; arrows, ≈21-nt small RNAs detected by miR167b antisense probe; diamond, ≈21-nt miR167b* small RNAs detected by the miR167b* antisense probe; D, DCL1; H, HYL1; S, SE.

Fig. 4.

Effects of HYL1 and SE on the processing accuracy of DCL1. (A–D) Distribution of sequenced small RNAs from in vitro processing reactions within that of the pri-miR167b substrate. Each small bar represents a single small RNA sequence. The recombinant proteins added to each reaction is indicated; n is the number of small RNAs sequenced from each reaction shown in Fig. 3B lanes 4, 7, 10, and 13. The positions of miR167b and miR167b* are indicated by black lines. (E) Processing accuracy. Small RNA products of DCL1 cleavage were cloned and sequenced. The accuracy of processing is defined as the percentage of sequences identical to miR167b or its complement miR167b* (precise) or identical sequences together with the fraction of sequences that are either offset by 1–2 nt or are 1- to 2-nt shorter than the miR167b sequence or its complement (almost precise). Incorrect cleavage is defined as the fraction of ≈21 -nt sequences derived from other parts of the pri-miR167b sequence, predominantly corresponding to its 3′end. I and II, in vivo processing accuracy or inaccuracy at MIR167b locus from Rajagopalan deep sequencing data (55) and ASRP database (54). The accuracy of in vivo processing is defined as representation of sequences identical to miR167b or its complement miR167b* as a fraction of all small RNA sequences derived from the MIR167b locus. Incorrect cleavage is defined as the fraction of ≈21-nt sequences derived from other parts of the MIR167b locus. D, DCL1; H, HYL1; S, SE.