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. 2014 Jul;20(7):1068-77.
doi: 10.1261/rna.044537.114. Epub 2014 May 22.

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

James M Burke  1 , Demetra P Kelenis  1 , Rodney P Kincaid  1 , Christopher S Sullivan  1
Affiliations

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

James M Burke et al. RNA. 2014 Jul.

Abstract

Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

Keywords: DGCR8; Drosha; RNAi; miRNA; pri-miRNA; shRNA.

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Figures

FIGURE 1.
FIGURE 1.
The distance from basal ssRNA of the SV40 pri-miRNA is only a partial determinant of Drosha cleavage. (A) Schematic diagram illustrating the mutagenesis strategy to generate the SV40 pri-miRNA variants. The basal ssRNA, internal loop, and/or the terminal loop positions of the SV40 pri-miRNA were increased (green), unchanged (blue), or decreased (red) in all combinations (detailed in Supplemental Fig. 1C). (B) Schematic diagram of the cellular-based Drosha–DGCR8 processing efficiency assay. (C) Renilla luciferase constructs containing either the K661 (pri-K661) or 776 (pri-WT) SV40 pri-miRNA were cotransfected with either pcDNA3.1+ or Drosha/DGCR8 expression vectors. The average luciferase activity ratio (Ren/FF) is graphed and normalized to pri-K661; error bars indicate SE (n = 6). (D) Schematic diagram of the base-anchor model (Han et al. 2006). DGCR8 (green) binds the basal ssRNA and directs Drosha (red) to cleave ∼11 bp up the stem. (E) Illustration of the Drosha cleavage sites of the indicated SV40 pri-miRNA variants determined by RNA seq (Supplemental Table S1) and Northern blot analysis. The large/green arrows and small/blue arrows represent the dominant and minor cleavage sites, respectively. The length of the arrows roughly correlates to the ratio of the derivative pre-miRNAs. The gray dotted lines indicate the basal and apical stem-ssRNA junctions of pri-S. The dotted blue line indicates the dominant cleavage site of pri-S. Alteration to the basal stems of pri-P and pri-T relative to pri-S are highlighted in blue. Each (+) represents a 20% increase in cleavage efficiency relative to pri-K661 (Supplemental Fig. S3). (F) Illustration of the cleavage site and efficiency of the indicated SV40 pri-miRNA variants as the basal stem was extended (highlighted in blue).
FIGURE 2.
FIGURE 2.
The terminal loop position of the SV40 pri-miRNA influences the Drosha cleavage position. (A) Schematic diagram of the loop-anchor model (Zeng et al. 2005). Drosha–DGCR8 (red) binds and directs cleavage ∼22 bp from the terminal loop. (B,C) Illustration of the cleavage sites and processing efficiency of indicated SV40 pri-miRNA variants when the apical stem was extended by 1 or 2 bp (highlighted).
FIGURE 3.
FIGURE 3.
The internal loop within the SV40 pri-miRNA stem influences cleavage site selection. (A) Illustration of the predicted secondary structure of pri-WT and a hypothetical variant (pri-NB) in which the internal loop was removed by pairing the bulged nucleotides in pri-WT (highlighted). (B) Three-dimensional model of pri-WT and pri-NB. (C,D) Illustration of the cleavage sites and processing efficiency for the indicated SV40 pri-miRNA variants as the internal loop (highlighted) is shifted +1 bp (arrow) up the stem (toward the loop). (E) Illustration of the cleavage sites and processing efficiency for the indicated SV40 pri-miRNA variants when 1 bp is removed from the apical stem while 1 bp is simultaneously inserted in the basal stem (highlighted).
FIGURE 4.
FIGURE 4.
The basal ssRNA, internal loop, and terminal loop cooperatively coordinate Drosha cleavage of the SV40 pri-miRNA. Illustration of the cleavage sites and processing efficiency for the indicated SV40 pri-miRNAs variants. (A) The apical and basal stem alterations relative to pri-R are indicated in pri-D, pri-K, and pri-O. The gray line indicates that the internal loop position is shifted +2 bp up the stem in the following pri-miRNA variants: pri-T, pri-F, pri-M, and pri-P. The basal and apical alterations relative to pri-T are indicated in pri-F, pri-M, and pri-P. (B) The internal loop position of pri-A is shifted up the stem +1 bp (pri-WT) and +2 bp (pri-B). The gray lines indicate 1-bp extension of the apical stem (pri-I) or a 1-bp deletion in the basal stem (pri-E) relative to pri-B.
FIGURE 5.
FIGURE 5.
The basal ssRNA and terminal loop coordinate the Drosha cleavage site of human pri-miRNAs. Illustration of the Drosha cleavage sites of pri-miR-30a and the indicated variants, as determined via a combination of RNA seq of the pre- and mature-miRNAs and Northern blot analysis of the pre-miRNAs. Alterations to the basal and apical stems are highlighted. (*) Only the pre-miRNA lengths, as determined by Northern blot analysis, were used to map the indicated cleavage site(s).
FIGURE 6.
FIGURE 6.
The apical- and basal-stem dimensions distinctly influence Drosha processing efficiency of the SV40 pri-miRNA. The secondary pri-miRNA structures for the indicated SV40 pri-miRNA variants (differences highlighted) are shown above a graphical representation of the processing efficiency for each pri-miRNA. Gray dashed lines represent the basal ssRNA-, internal loop-, and terminal loop-junctions. Bar graphs depict the mean relative luciferase activity (Ren/FF); error bars represent the SE (n = 6).
FIGURE 7.
FIGURE 7.
Model of pri-miRNA recognition and processing by Drosha–DGCR8. Illustration of the ssRNA-cooperation model for Drosha–DGCR8 recognition and processing of pri-miRNAs. The dashed gray lines represent ssRNA/dsRNA junctions. The dashed red arrows represent the influence from the indicated structures on Drosha cleavage site selection (black arrows). Drosha/DGCR8 (light green); DGCR8 dsRBDs (dark green); hypothetical loop recognition factor (blue); hypothetical stem cofactor (orange); hypothetical basal ssRNA recognition factor (yellow).
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