Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA

Abstract

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.

Figure 1.

Overview of patient-derived Dicer mutations located within the Platfrom-PAZ domain. (A) Table of patient-derived alterations with corresponding phenotypes. (B) Three-dimensional representation of the Platform-PAZ domain (Protein Data Bank entry 5ZAK) with the location of patient-derived mutations highlighted and close-up views of the secondary structural features surrounding S839 and L881. Residues associated with point mutations are shown as ball and stick models, while frame shift and deletion mutants are grouped by large dashed and solid boxes, respectively. Major RNA-binding pockets are highlighted in light blue with key amino acids colored gray. α-Helices 1 and 2 are colored gray in all models.

Figure 2.

Characterization of the binding and cleavage activity of WT and Dicer mutants for pre-miR-21 in vitro and in cells. (A) Representative EMSA data (Figure S3) shown over the course of 120 min. (B) Quantification of binding at the 120 min time point as determined using the relative difference in the intensity of bound RNA (top band) to unbound RNA (bottom band). (C) Representative time-dependent cleavage visualized via gel electrophoresis (Figure S4). (D) Quantification of Dicer cleavage activity determined on the basis of the relative changes in the band intensity of the uncleaved RNA (top) band and the cleaved RNA (bottom) bands. Bands between these regions, which represent intermediate cleavage products, were not included in our quantification. Gel-based analysis was carried out using ImageJ. Error bars represent the standard deviation from three independent experiments (Tables S1 and S2). (E and F) Impact of Dicer mutations on miR-21 and Dicer protein levels in Dicer1−/− cells. (E) miR-21 expression in WT and Dicer mutant-transfected cells from three biological replicates (Figure S6 and Table S3). RNA levels were normalized to U6 snRNA, and fold changes in miR-21 expression were calculated relative to WT Dicer. (F) Relative protein levels of WT and Platform-PAZ Dicer mutants from three biological replicates (Figure S6 and Table S4). Protein levels were quantified through densitometry analysis from Western blots using ImageJ. Mutant protein levels were normalized to WT Dicer. The variability of the cell-based assays is likely due to deviations in transfection efficiency (Figure S5).

Figure 3.

Characterization of the binding and cleavage activity of WT and Dicer mutants for snord37. (A) Predicted secondary structures of pre-miR-21 and snord37 as determined via RNAfold 2.4.18. The color scale indicates base-pair probabilities. (B) Representative EMSA data from three independent experiments (Figure S3) shown over the course of 120 min. (C) Quantification of binding at the 120 min time point as determined using the relative difference in intensity of bound RNA (top band) to unbound RNA (bottom band). (D) Comparison of binding to pre-miR-21 and snord37 at 120 min. (E) Quantification of time-dependent Dicer cleavage activity (Figure S4). Gel-based analysis was carried out using ImageJ. Error bars represent the standard deviation from three independent experiments (Tables S1 and S2). (F) Comparison of cleavage of pre-miR-21 and snord37 at 120 min by Dicer disease mutants S839F and L881P.

Figure 4.

Comparison of the binding, cleavage activity, and cleavage product formation of WT and Dicer mutants for pre-miR-21, snord37, and blunt pre-miR-21. (A) Comparison of binding across substrates at 120 min. Quantified data from three independent experiments (Figure S3 and Table S1). (B) Characterization of Dicer cleavage product formation; representative data from three independent experiments (Figure S4). Cleavage products were observed for pre-miR-21 and snord37 after a 120 min reaction. Blunt pre-miR-21 showed an increase in the levels of multiple cleavage products of varying sizes based on positions in the gel.