Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis

Abstract

Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Export in 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3' end, yielding miRNAs from the 5' strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.

Keywords: DICER; DROSHA; Exportin 5; knockout; microRNA.

Fig. 1.

Nuclease-mediated knockout of miRNA maturation factors. (A) Domain structure of DROSHA, XPO5, and DICER proteins. Red triangles indicate the regions corresponding to the genomic DNA sequences that are targeted by nuclease. For the knockout of DROSHA and XPO5, a single genomic DNA region was selected to be targeted by Cas9 and TALEN, respectively. In the case of DICER, two guide RNAs were used to cleave the adjacent genomic regions. (B) Targeted genomic sequences. Red letters in DROSHA and DICER indicate the regions recognized by guide RNAs. Red letters in XPO5 indicate the region targeted to be cleaved by TALEN. Italic and underlined letters show insertion, whereas green letters stand for substitution. Blue letters in DROSHA and DICER indicate the Protospacer Adjacent Motif (PAM) recognized by Cas9 protein. Blue letters in XPO5 indicate the binding region of TALEN pairs. On the right side, the number of mutated nucleotides and the sequencing frequency of the allele in the cell clone are presented. (C) Western blot experiments to confirm gene disruption. (D) Proliferation of parental and knockout cells measured by cell counting. Error bars show deviation from two independent experiments.

Fig. S1.

(A–D) Uncropped Western blot image to confirm the ablation of targeted gene in each knockout. The information of antibody used for each experiment is indicated below the blot. The data for DICER in D is a whole blot image of the same blot presented in the Fig. 1C. The asterisk in B indicates a nonspecific band. (E) Western blot to measure the level of miRNA biogenesis factors. Note that the levels of the biogenesis factors other than the targeted one were not affected in the knockout, except for the DICER level in DROSHA and XPO5 knockout cells.

Fig. 2.

Global analysis of miRNA expression in knockout cells. (A) The proportion of miRNA reads in the small RNA sequencing libraries from knockout cells and their parental cells. Two libraries from independent samples were generated from DROSHA knockout cells and its corresponding parental cell line (Left). As for XPO5 and DICER, one library was made from each knockout clone (Right). (B-D) Expression change of miRNAs after the knockout was depicted by scatter plot. The top 200 miRNAs based on their sequencing reads in parental cells were selected for the analysis. For DROSHA knockout (B), normalized fold changes between two replicates were compared. For XPO5 (C) and DICER knockouts (D), normalized fold changes between two different knockout clones were compared. The miR-320a-3p level was used for normalization in DROSHA and XPO5 knockout sets. For the DICER knockout set, those reads aligned to rRNAs and tRNAs were used for normalization. Outliers and those that are validated by Northern blot are indicated. (E) Based on the fold change, the number of miRNAs reduced after the knockout were counted.

Fig. S2.

(A–C) The abundance ranks of miRNAs were compared between the parental and DROSHA knockout (A), XPO5 knockout (B), and DICER knockout cells (C), respectively. The miRNAs were ranked based on the read numbers in Dataset S1. In A, DROSHA-dependent and -independent miRNAs were marked with different colors, respectively. (D–F) The same miRNAs in Figs. 2 B–D were depicted based on their sum of sequencing reads of parental and knockout libraries (x axis), and their fold changes after the knockout (y axis).

Fig. 3.

miRNA expression in the DROSHA knockout cells. (A) Northern blot analysis to validate the changes in miRNA levels in the knockout. Canonical miRNAs such as miR-16-5p and miR-21-5p are not detectable, whereas noncanonical miRNAs including miR-320a-3p and miR-484–3p were readily observed. The dashed lines indicate discontinuous lanes from a single gel, which is true for all of the figures with dashed lines throughout this paper. (B) List of miRNAs produced in the DROSHA knockout cells. Bold letters indicate those that are newly identified DROSHA-independent miRNAs in this study. Those miRNAs with a fold change larger than 0.01 (DROSHA knockout/parental) are shown. A table for top 200 miRNAs based on the sequencing reads in parental cells is provided in Dataset S1. According to the sequencing reads and gene structure, miRNAs were classified into 5′ capped miRNA, mirtron, or endo-siRNA. (C) Predicted secondary structure of pre-miR-7706 and the sequencing data in comparison with the AKAP13 gene structure. The CAGE data from ENCODE project were obtained from UCSC genome browser (genome.ucsc.edu/). The sequencing results combined from two libraries were used to depict the graph showing the reads at each nucleotide position. The y axis of the graph was normalized based on the level of miR-320a-3p, a DROSHA-independent miRNA. The miRNA sequence produced from 3p strand is indicated with yellow shade. Based on the sequencing data and the structure of the host gene, the secondary structure of pre-miR-7706 was predicted and shown on the right. Note that the 5′ end nucleotide (indicated with a red letter) of pre-miR-7706 coincides with the transcription start site of AKAP13 isoform (BP871581), a probable host gene of miR-7706. Because there is no CAGE data available for colorectal cancer cell lines including HCT116, we analyzed the CAGE data from other randomly selected cell lines, GM12878, A549, and IMP90. All of the cell lines included in the ENCODE project showed similar CAGE patterns. The same analysis as in C was applied to D miR-3615. In addition to the RefSeq mRNA SLC9A3R1 (NM_004252), an mRNA from GenBank, AK026581, was also shown in D because the latter is expected to be a more probable host gene of miR-3615 based on its 5′ end sequence.

Fig. S3.

(A) A predicted secondary structure of miR-484, nearby genomic information, and sequencing results are shown. The same analysis as in Figs. 3 C and D was applied. Based on the structure of the host gene and the sequencing data, pre-miR-484 was predicted and shown on the left. Because the sequence of miR-484 hairpin is mis-annotated in miRBase, an alternative secondary structure is depicted. In this alternative structure, the 5′ end nucleotide (indicated with red letter) of pre-miR-484 coincides with the transcription start site of the NDE1 gene (NM_001143979). Note that the CAGE signal is not observed at the 5′ end of NM_001143979, but observed at the 5′ end of NM_017668. (B) Genomic information near pre-miR-1254-1. The genomic information of CCAR1 and Alu elements was obtained from UCSC genome browser (genome.ucsc.edu/). The genomic location of pre-miR-1254-1 was obtained from miRBase annotation (mirbase.org/), while that of miR-1254-5p was analyzed from the sequencing data. Note that the position of pre-miR-1254-1 overlaps with that of Alu sequence. The secondary structure of AluJr containing miR-1254-5p (indicated with yellow color) was predicted from mfold web server (unafold.rna.albany.edu/?q=mfold). Although there is another member in the miR-1254 family, that is pre-miR-1254-2, we only selected the pre-miR-1254-1 for the analysis because no transcriptional activity was observed near pre-miR-1254-2 in the parental and DROSHA knockout cells.

Fig. 4.

miRNA expression in the XPO5 knockout cells. (A) The expression of miRNAs from parental cells and XPO5 knockout cells was measured. (B) The expression level of miRNAs in the nucleus and cytoplasm of parental cells and XPO5 knockout cells, respectively, was compared. (C) The change in expression level after XPO5 knockout was compared between the miRNAs from 5p and 3p strands. Only the reads with perfect match to miRNA sequences were selected for analysis. P value was calculated by two-sided Wilcoxon rank-sum test. (D) The change in length after XPO5 knockout was compared between the miRNAs from 5p and 3p strands. P value was calculated by two-sided Wilcoxon rank-sum test. (E) The proportion change in miRNAs that are trimmed or added after XPO5 knockout was analyzed at each end of the miRNA sequences. The proportions of miRNAs with sizes less than those of reference sequences were analyzed using the sequencing data from parental cells and XPO5 knockout cells, respectively, and their differences were calculated to be used as the change in “Trimmed” proportion. For the analysis of 5′ end, only the miRNA reads whose 3′ end coincides with the 3′ end of reference sequence were used, and vice versa. The same analysis was applied to calculate the proportion change of “Added” miRNA except that longer sequences than reference sequence were collected for the analysis. P value was calculated by two-sided Wilcoxon rank-sum test.

Fig. 5.

miRNA expression in the DICER knockout cells. (A) The expression of miRNAs in parental cells and DICER knockout cells was measured. Note the bands for miRNA fragments between pre-miRNA and mature miRNA. (B) Change in the length of 5p strand miRNAs was compared between parental and DICER knockout cells. P value was calculated by two-sided Wilcoxon rank-sum test. (C) Change in the expression level of miRNAs was compared between 5p and 3p strand miRNAs. P value was calculated by two-sided Wilcoxon rank-sum test. (D) The expression of individual miRNA was compared between the sequencing libraries made with Dicer heterozygous knockout and null knockout mouse cells, respectively (35). The miRNAs produced from 5p and 3p strands are shown with different colors. (E) The change in expression level of miRNAs produced from 5p and 3p strands were compared. (F) The length change of 5p strand miRNAs were compared between Dicer heterozygous knockout and null knockout cells. The same data in D were used for the analysis in E and F. P value was calculated by two-sided Wilcoxon rank-sum test. (G) Association of 3′ extended-5p miRNAs with AGO proteins. Input RNA was prepared from 200 µg of protein extracts. For the immunoprecipitation of AGO proteins, 2.4 mg of protein extract was used. The associated miRNAs with AGO proteins were measured by Northern blot. (H) Model for the generation of 3′ extended-5p miRNAs in the absence of DICER. Although pre-miRNAs are loaded into AGO proteins, their 3′ ends may be more vulnerable to nuclease attack if they are not processed by DICER rapidly. The nuclease may trim the pre-miRNA until most of the terminal loop is removed, but further trimming might be hindered by Ago proteins making 3′ extended-5p miRNAs.

Fig. S4.

(A) The sequencing reads aligned to miR-16-2 and miR-10b in the parental or DICER knockout library are shown as a graph. The y axis of the graph was normalized based on the reads aligned to rRNAs and tRNAs. Mature miRNA sequences are indicated with yellow shades. Note the increased proportions of reads of 3′ extended-miRNA from 5p strand in the DICER knockout. (B) The expression of individual miRNA was compared between the sequencing libraries made with wild type and dicer mutant zebrafish embryo, respectively (24). Only the pre-miRNAs producing both 5p and 3p strands miRNAs were selected for the analysis. The miRNAs produced from 5p and 3p strands are shown with different colors. (C) The change in expression level of miRNAs produced from 5p and 3p strands were compared. (D) The length change of miRNAs produced from 5p and 3p strands were compared. The same data in B was used for the analysis in C and D. P value was calculated by one-sided paired Wilcoxon rank-sum test.

Fig. 6.

Biogenesis pathways of miRNAs. The miRNAs are categorized into six groups based on the requirement for each biogenesis factor. Nuclear export of pre-miRNAs can be mediated by the factor other than XPO5 or XPO1, although not indicated in this figure. This figure was modified from ref. . Please refer to the text for a detailed description.