Ensemble analysis of primary microRNA structure reveals an extensive capacity to deform near the Drosha cleavage site

Abstract

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary microRNAs, revealing deviations from canonical double-stranded RNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by noncanonical structure elements, combine with the necessary single-stranded RNA-double-stranded RNA junction to define the correct Drosha cleavage site.

Figure 1

SHAPE-constrained MC-Fold calculations yield secondary structures with embedded estimation of conformational dynamics. SHAPE reactivity traces (left) identify single-stranded nucleotides for (A) pri-mir-16-1, (B) pri-mir-30a, and (C) pri-mir-107. In the SHAPE reactivity traces, bar heights indicate the normalized mean reactivity constructed from 21 independent reactions. Blue filled bars indicate nucleotides with a positive mean reactivity and a magnitude greater than the uncertainty of the measurement. All grey bars indicate that the reactivity is either negative or has a mean magnitude below the uncertainty and are therefore considered insignificant. Addition of SHAPE-derived single-stranded constraints to MC-Fold calculations yields the combined probability of the nucleotide being single-stranded, which is mapped onto the most probable secondary structure (right). These probabilities are indicated in color as annotated in the color bar, ranging from most likely double-stranded (blue) to most likely single-stranded (red). Regions of high single-stranded probability divide the stems into three segments, labeled as H1, H2, and H3. Nucleotide numbering corresponds with the pri-miRNA numbering starting at 1 and the SHAPE cassette linkers (see Materials and Methods) being less than 1 and greater than the pri-miRNA length. Nucleotides corresponding to the mature miRNAs (as annotated in miRBase) are indicated by a line adjacent to the secondary structure diagram, which has been oriented such that the Drosha cut site is on the left in all three cases. Secondary. structure diagrams were generated in VARNA (54).

Figure 2

Ribonuclease structure mapping is consistent with the most probable secondary structure resulting from the SHAPE-constrained MC-Fold calculations for (A) pri-mir-16-1, (B) pri-mir-30a, and (C) pri-mir-107. For each RNA, a denaturing 12% polyacrylamide gel used in the analysis is shown, with lanes as follows: C is a control sample (no nuclease); OH⁻ is a limited alkaline digest; and T1, A, and V1 are limited digests with ribonucleases specific for single-stranded G, single-stranded C and U, and 5′ to double-stranded or well stacked single-stranded regions, respectively. The reactions in lanes 2 and 3 were performed under RNA-denaturing conditions (denoted ‘Den.’) in order to provide a ladder correlating position in the gel with the nucleotide sequence; while the reactions in lanes 4–6 were performed under RNA-native conditions (denoted ‘Nat.’). Helical and loop regions of the RNA are indicated to the right of the gel. The highest probability secondary structure (see Fig. 1) with positions of cleavage by ribonucleases under native conditions indicated by symbols as described in the legend is displayed below each gel. Symbol size is proportional to cleavage intensity. In these secondary structure maps, proposed Drosha cleavage sites are identified with red arrows; regions near Drosha cleavage sites displaying high single-strand probability in our MC-Fold and SHAPE analysis are enclosed in grey boxes.

Figure 3

Ensemble representation of the top five SHAPE-constrained models generated by MC-Sym for (A) pri-mir-16-1, (B) pri-mir-30a, and (C) pri-mir-107. All models are aligned along the main stem of the RNA with the sugar-phosphate backbone indicated by a blue ribbon. Planks representing the nucleotides are colored according to the probability of being single-stranded as reported in Figure 1 and indicated by the color bar, ranging from most likely double-stranded (blue) to most likely single-stranded (red). Regions of high single-stranded probability divide the stems into three segments, labeled as H1, H2, and H3. Inclusion of extended single-stranded tails renders MC-Sym calculations unstable; therefore, the expected tails are not represented in the models shown.

Figure 4

The secondary structures of pri-miRNA molecules harbor multiple dynamic bulges and internal loops. Expanded views of areas within the MC-Sym models that are highly dynamic are shown for (A) A/A mismatch in pri-mir-16-1, (B) U bulge in pri-mir-16-1, (C) CU bulge in pri-mir-30a, and (D) 1-by-3 asymmetric internal loop in pri-mir-107. The nucleotides involved in the imperfections are colored orange (their position in the nucleotide sequence is also annotated) and the most probable structure is shown otherwise in solid blue, with the models from the other four members of the ensemble reported in Figure 3 shown in transparent blue. All models are aligned to the nearest stable Watson-Crick base-pair neighboring the imperfection in the most probable model.

Figure 5

The secondary structures of pri-miRNA molecules harbor multiple non-Watson-Crick mismatches that are predicted to be well-ordered by SHAPE reactivity. Expanded views of areas within the MC-Sym models representing these mismatches are shown for (A) A/C mismatch in pri-mir-30a, (B) U/C mismatch in pri-mir-107, (C) C/U mismatch in pri-mir-16-1, and (D) AG•GA internal loop in pri-mir-16-1. The nucleotides involved in the imperfections are colored orange (their position in the nucleotide sequence is also annotated) and the most probable structure is shown otherwise in solid blue, with the models from the other four members of the ensemble reported in Figure 3 shown in transparent blue. All models are aligned to the nearest stable Watson-Crick base-pair neighboring the imperfection in the most probable model.

Figure 6

Drosha processing of pri-miRNAs to pre-miRNAs in vitro confirms the necessity of hot spot flexibility for efficient cleavage. (A) The mutant pri-mir-16-1-HS has significantly reduced hot spot flexibility compared with wild-type, as established by combined SHAPE and MC-Fold analysis. (B) The mutant pri-mir-107-HS has the asymmetric 1-by-3 internal loop near the Drosha cleavage site replaced by a flexible C•C non-canonical base-pair, (C) while the mutant pri-mir-107-HS2 has only Watson-Crick base-pairs at the cleavage site, as established in both cases by combined SHAPE and MC-Fold analysis. (D) Denaturing gels for the processing of (from left to right) pri-mir-16-1 wild-type (WT), hot spot mutant (HS mut), and tetraloop mutant (TL mut) constructs; in addition to pri-mir-107 wild-type (WT), hot spot mutant (HS mut), and second hot spot mutant (HS mut2) constructs. In all six assays, lanes represent RNA collected prior to addition of Microprocessor (RNA), exposed to FLAG-beads with addition of cell lysate that did not express FLAG-tagged proteins for 5 minutes (Mock), exposed to GFP for 5 minutes (GFP), and 5 minutes after exposure to purified Microprocessor (Micro.). (E) The percentage of pri-miRNAs cleaved by the Microprocessor in vitro after 5 minutes averaged over three independent experiments. Cleavage is calculated as the sum of the intensities of the pre-miRNA product and the cleaved flanking tails divided by the sum of the intensities of the product, tails, and the remaining pri-miRNA substrate.