Lower and upper stem–single-stranded RNA junctions together determine the Drosha cleavage site

Significance

MicroRNA genes are transcribed as long primary microRNAs (pri-miRNAs) and then processed by the Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex into ∼60-nt pre-miRNAs, which is the key step of miRNA biogenesis. How exactly the Drosha–DGCR8 complex recognizes pri-miRNAs and selects the processing site is not well understood. Here we developed a unique approach to examining the details of how this complex chooses the processing site in human cells. Our study yields new knowledge of how the Drosha–DGCR8 complex determines its processing sites and also provides an explanation for how 5′ isomiRs are generated in some miRNAs, which is another interesting but not clearly understood phenomenon in miRNA biogenesis.

Abstract

Microprocessor [Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha–DGCR8 complex measures either ∼22 nt from the upper stem–single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem–ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem–ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5′ isomiRs. Thus, our results also reveal a mechanism of 5′ isomiR generation.

In mammals, the canonical pathway of miRNA biogenesis is initiated by the Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex (the Microprocessor), which processes long primary miRNAs (pri-miRNAs) into ∼60-nt pre-miRNAs for further processing by Dicer into a duplex ∼22 nt long. One or both strands of the duplex are loaded into the RNA-induced silencing complex (RISC) to repress target gene expression (1, 2). Although the major players of miRNA biogenesis are mostly known, no model precisely predicts how the miRNA biogenesis machinery recognizes and processes a novel pri-miRNA or an miRNA-mimicking shRNA and which strand will finally be loaded into RISC. The missing details in miRNA biogenesis are major obstacles to rational design of miRNA-based shRNA or artificial miRNA (amiRNA) that generates predictable results, as this kind of amiRNA must be processed by the endogenous miRNA machinery to be functional. The lack of a precise model of miRNA biogenesis also makes it difficult to accurately predict novel miRNA genes in the genome. In fact, a recent study found that more than 150 miRNAs annotated in miRBase (of 564 miRNAs checked) were not miRNAs at all (3). Thus, there is still a need to improve our understanding of how the miRNA biogenesis machinery recognizes and processes pri-miRNA into mature miRNA.

The secondary structure of a canonical pri-miRNA usually consists of four parts: a terminal loop, an upper stem that encompasses the mature miRNA duplex of approximately two helical turns, the lower stem, which is an approximate one-helical-turn extension of the miRNA duplex, and the basal segments, which are single-stranded flanking sequences (4, 5). In 2005, Zeng et al. proposed that a large terminal loop is required to guide Drosha cleavage to occur at ∼22 nt from the junction of the terminal loop single-stranded RNA (ssRNA) and upper stem (5). In addition, they showed that single-stranded flanking sequences around the miRNA hairpin structure are also required for efficient Drosha processing (6). However, in 2006, Han et al. proposed an alternative model in which the major component of the Microprocessor, DGCR8, recognizes the lower stem–ssRNA (basal segment) junction, guiding Drosha to cut ∼11 nt from the junction, although they also acknowledged that the terminal loop region might also be recognized by the Microprocessor (4). A recent report from Zeng’s laboratory reinforced their previous conclusion that the terminal loop is critical for Microprocessor processing (7). Auyeung et al. recently analyzed a large number of variants derived from four different miRNAs and identified three primary sequences that are critical for Microprocessor recognition of pri-miRNA. In addition, they showed that, although the terminal loop region contributes to recognition and processing for some pri-miRNAs, it appears to be less important than the lower stem region (8). However, how the lower stem and terminal loop region coordinate selection of the Drosha processing site is not clear.

Another interesting question in miRNA biogenesis concerns the end-heterogeneity of mature miRNAs. The end of mature miRNAs is not absolutely fixed, but subject to variation (9–12), and we and other groups have previously discovered the pattern of these variations (13, 14). Although 3′ end variation is a widespread phenomenon, the 5′ end of mature miRNA is highly uniform for most miRNAs. However, in some miRNAs, such as miR-142, the 5′ end is also highly variable (13). These 5′ isomiRs are of particular interest because positions 2–7 of the 5′ end of miRNAs have been called the “seed sequence,” which determines the target pool of the miRNA (15). Although 5′ isomiRs have only a one- or two-nucleotide difference in their 5′ end, they have different seed sequences and different pools of target genes, as shown in a recent report (16). Thus, 5′ isomiRs appear to be a very efficient way for an miRNA to expand its pool of target genes. To understand why 5′ isomiRs occur in some miRNAs but not in others, we previously cloned pre-miRNAs generated in human cells and shown that in most miRNAs that have highly uniform 5′ ends, their pri-miRNAs are processed precisely at one site to generate one pre-miRNA, which gives rise to one or two (in the case of both strands of the duplex being loaded into RISC) dominant mature miRNAs. By contrast, in some other miRNAs, such as miR-142, that have a high frequency of 5′ end variation, the pri-miRNAs are cleaved at different sites to generate multiple distinct pre-miRNAs, which give rise to multiple 5′ isomiRs (17). Thus, 5′ isomiRs are caused by alternative Drosha processing in some miRNAs, such as miR-142. A recent report showed that 5′ isomiRs can also be generated by Dicer partner protein binding in some other miRNAs, such as miR-132 (16). Why alternative Drosha processing occurs only in some miRNAs but not in others has not been clear.

Here we made a series of mutations to various pri-miRNAs to change the distance from the lower stem–ssRNA junction and/or upper stem–ssRNA junction to the original processing site and determined the Drosha processing site in human cells. Our data show that Drosha cleavage is coordinated by both the lower and upper stem–ssRNA junctions. Reduced coordination by altering the stem length by 1 or 2 bp in either direction causes alternative Drosha processing in which Drosha alternatively cleaves at multiple sites and generates multiple 5′ isomiRs.

Results

miRNA-Offset RNA Can Be Used to Reliably Determine Drosha Cleavage Sites.

miRNA-offset RNAs (moRs) were first described in Ciona intestinalis and described as a class of small RNAs ∼20 nt long and immediately adjacent to a pre-miRNA (18). It has been proposed that moRs might be the byproducts of Drosha cleavage, and the end of the moR might indicate the Drosha processing site (18, 19). We analyzed the small RNAs cloned from 293FT cells to see whether it is feasible to use moRs to determine the Drosha cleavage site. In a few miRNAs, for example, miR-18a, the moRs were cloned in small numbers and usually precisely indicate the Drosha cleavage site. However, for most miRNAs, such as miR-16, miR-17, and miR-92a, which are three of the most abundant miRNAs in 293FT cells, not even a single read of moR sequence was cloned, although the mature miRNAs yielded more than 30,000 reads each (Dataset S1), which is consistent with a previous report that moRs are not abundant in human cells (19). This fact dampened our enthusiasm for using moRs to determine Drosha cleavage sites in human cells. However, a recent finding from our study on miRNA-based shRNAs prompted us to believe that moRs could be used to reliably identify Drosha processing sites in human cells. Previously, we designed two amiRNAs in which the mature miRNA duplex sequence was replaced by an siRNA duplex, and transcription was driven by the strong pol III promoter U6. The constructs were transfected into 293FT cells and the small RNA products were cloned to check how exactly these amiRNAs are processed in human cells. Interestingly, in addition to mature siRNA sequences, large numbers of moR sequences were also cloned, especially 5′ moRs. The 3′ end of the 5′ moR and the 5′ end of the 3′ moR were highly uniform (Datasets S2–S4). The ends could be perfectly aligned with representative mature products of the amiRNA, and these ends formed a perfect 2-nt overhang, which is a typical structure generated by an RNase III enzyme like Drosha (Fig. S1 A and B), suggesting that the ends perfectly indicate the exact Drosha cleavage site in human cells.

Encouraged by these results, we transfected pri-miR-150 and -122 into 293FT cells, which do not express these miRNAs endogenously, and cloned the small RNA products. Consistent with the amiRNA experiments, large numbers of moR sequence reads were cloned, and the ends appear to indicate the exact Drosha cleavage site (Fig. S1 C and D). In all four cases, the 3′ end of the 5′ moR, the 5′ end of the 3′ moR, and the 5′ end of the 5′ arm mature species appear to be highly uniform and perfectly indicate the exact Drosha cleavage site (Fig. S1 A–D). The 3′ ends of the 3′ arm mature species can be varied dramatically, as seen in miR-122 (Fig. S1D), which is not surprising, as it is well known that the 3′ ends of mature miRNAs are under active modification (13, 20–23). Thus, to avoid confusion, the 3′ end of the 3′ arm of mature species will not be used to identify Drosha cleavage sites in the following experiments. It is noteworthy that the 3′ end of the 5′ moRs appears to be the most uniform end (Fig. S1 A–D), suggesting that the 3′ end of the 5′ moRs provides a clearer picture of where Drosha cleaves.

To ensure the accuracy of moR prediction of Drosha processing, we also cloned the ends of the pre-miRNAs for miR-150 and miR-122. Unlike mature miRNAs, which go through multiple steps after Drosha processing, pre-miRNAs are the direct products of Drosha cleavage and therefore should be better at indicating the Drosha cleavage site than mature miRNAs. However, the 3′ end of pre-miRNAs is also subject to extensive modification (3, 17, 24); thus, we chose to clone the 5′ end of pre-miRNAs to determine the Drosha cleavage site. Despite the high noise, the results are consistent with the results for moRs and mature miRNAs (pre-miR-150 and pre-miR-122 in Table S1). These data further support our hypothesis that moRs can be used to identify the Drosha cleavage site in human cells.

Previously, it has been reported that two hairpin structures of DGCR8 mRNA, hairpin A and hairpin B, can be processed by the Drosha–DGCR8 complex in vitro, but no mature miRNA sequences were detected from these two hairpins in cells (25, 26). A pre-miRNA-like product could be detected for hairpin A by Northern blot in human cells, suggesting that the hairpin could be processed by the Drosha–DGCR8 complex in human cells (26). Thus, we chose hairpin A to determine whether moRs can be used for identifying the Drosha cleavage site in this context. The construct harboring hairpin A was transfected into 293FT cells, and the small RNAs were cloned. As expected, no mature miRNA products were generated from the hairpin. Most of the reads that were cloned were 5′ moRs, whereas no 3′ moR reads were cloned (Fig. S1E). According to the previous report, the Drosha–DGCR8 complex can process hairpin A at site A1 or site A2 in vitro; however, only one band that correlates with site A2 has been detected in human cells, suggesting that site A2 might be the Drosha cleavage site in human cells (26). Indeed, most reads cloned from the transfected hairpin A indicated that Drosha cleaved at site A2 (Fig. S1E). The data therefore suggest that 5′ moR sequences can be used to predict Drosha cleavage site of mRNAs, even when they do not generate miRNAs. Thus, we will mainly use 5′ moRs in combination with 3′ moRs and the 5′ arm mature miRNAs to identify Drosha cleavage sites in this study.

Extending the Lower Stem Causes Alternative Drosha Processing.

To understand how the lower stem–ssRNA junction regulates Drosha processing, we constructed a series of mutations of the pri-miR-150 with altered distances from the lower stem–ssRNA junction to the Drosha cleavage site. We started by mutating one nucleotide in the lower stem to increase the distance between the lower stem–ssRNA junction and the original Drosha cleavage site by 1 bp (miR-150 LS+1 in Fig. 1A). According to Han et al.’s model (4), this change should shift the Drosha cleavage site toward the lower stem–ssRNA junction by 1 bp. Interestingly, this single mutation instead caused alternative Drosha processing in which Drosha cuts at the original site (site 1) and also at a site 2 nt upstream (site 2), judging from the 5′ moR sequences (Fig. 1A and Table S2). Consistent with the 5′ moR results, two dominant mature miR-150 5′ isomiRs corresponding to two Drosha cleavage sites were cloned (Fig. S2A and Table S2), supporting the idea that Drosha indeed cuts at these two sites.

Fig. 1.

Increasing the distance between the lower stem–ssRNA junction and the Drosha cleavage site causes alternative Drosha processing. (A) A single mutation from C to G that extends the lower stem by 1 bp in pri-miR-150. Mature miRNA sequences are highlighted red. The mutations are indicated in green. Representative reads of cloned small RNAs are found in Table S2. Arrows represent the Drosha cleavage sites, as judged from the moRs. The length of the arrows represents roughly the frequency of moRs. The dotted line indicates the position of the original dominant Drosha cleavage site. (B) miR-150 LS+2, with mutations that extend the lower stem by 2 bp. (C) miR-150 LS+A, with insertion of 1 bp in the middle of the lower stem. (D) miR-150 LS+AU, with insertion of 2 bp in the middle of the lower stem. (E) Cloned pre-miRNA 5′ ends generated from mutated pri-miR-150s. Only the dominant reads are presented. The raw data are found in Table S1. (F) Northern blot of pre-miRNAs generated from mutated pri-miR-150s. (G) miR-122 LS+U, with a 1-bp insertion. (H) miR-122 LS+UG, with a 2-bp insertion.

Extending the lower stem by 2 bp (miR-150 LS+2) also caused alternative Drosha processing that cuts at the same sites as miR-150 LS+1, but it appeared that extending the lower stem by 2 bp induced Drosha to cut at a higher frequency at site 2 (63.7%) than at site 1 (35.5%), judging from the 5′ moRs (Fig. 1B and Table S2). The mature miR-150 isomiRs corresponding to site 2 (72.6%) were cloned at much higher frequency than those corresponding to site 1 (24.4%), suggesting that Drosha indeed cuts more at site 2 in miR-150 LS+2 (Fig. S2B and Table S2). Overall, it appears that extending the lower stem by 1 or 2 bp does not move the Drosha cleavage site 1 or 2 nucleotides toward the lower stem–ssRNA junction but instead causes alternative Drosha processing in which Drosha cleaves at two sites alternatively and generates two 5′ isomiRs. To further confirm that the alternative Drosha processing was caused by the distance change from the lower stem–ssRNA junction to the original Drosha processing site, we extended the lower stem by inserting 1 bp (miR-150 LS+A) or 2 bp (miR-150 LS+AU), as shown in Fig. 1 C and D. Although the pattern was slightly different, extending the lower stem by inserting 1 or 2 bp also caused alternative Drosha processing, just like miR-150 LS+1 and miR-150 LS+2 (Fig. 1 C and D and Fig. S2 C and D). These results further support the idea that extending the lower stem causes alternative Drosha processing.

The number of 3′ moR reads in this set of data did not correlate well with the number of 5′ moRs and the mature miRNA data, although the cleavage sites were mostly well correlated (Fig. 1 A–D and Fig. S2 A–D). Therefore, to confirm the Drosha cleavage site, we also cloned the 5′ end of the pre-miRNA for these mutated miRNAs. In all four mutations, two dominant pre-miRNA isoforms were cloned, and the number roughly correlated with the number of 5′ moR reads and the mature miR-150 data (Fig. 1E). We also performed a Northern blot to detect pre-miRNAs generated from the mutations. As shown in Fig. 1F, by comparing with WT miR-150, which has one dominant band, all mutations showed an extra band that is ∼4 nt longer. Moreover, the density of bands roughly correlated with the number of 5′ moRs and the mature miRNA data. The pre-miRNA data supported the idea that Drosha indeed cleaves at the sites predicted from 5′ moRs and mature miRNAs to generate two major pre-miRNAs. Thus, it appears that the 3′ moR is not a reliable indicator for determining the Drosha processing site. In addition, the number of cloned 3′ moRs is generally much fewer than the number of 5′ moRs, which, consistent with previous reports (18, 19), makes using 3′ moR to determine the cleavage site more vulnerable to experimental noise. We believe that this might be the reason for the inconsistency between 3′ moR and 5′ moR results here and also in several cases in the following experiments.

To test whether extending the lower stem causes alternative Drosha processing is a general phenomenon or occurs only in miR-150, we repeated the experiments with miR-122. We inserted 1 or 2 bp in the lower stem of the pri-miR-122, transfected the constructs into cells, and cloned the small RNA products. The moR results were consistent with the results for miR-150, where inserting 1 bp (miR-122 LS+U) caused Drosha to alternatively cut at the original site (site 1, 40.6%) and at a site 2 nt upstream (site 2, 56.1%), whereas inserting 2 bp (miR-122 LS+UG) caused Drosha to cut more at site 2 (67.8%) than at site 1 (31.1%) (Fig. 1 G and H and Table S2). However, although the moR results show that the Drosha cleavage site is localized more at site 2, very few reads of mature products were generated from this cleavage site—more than 200-fold fewer reads than for the native pre-miR-122 in both mutants (Fig. S2 E and F and Table S2). The reason could be that the pre-miRNA generated from site 2 could not go through the processing steps, including transporting, Dicer processing, and loading, as efficiently as the original pre-miR-122 generated from site 1. Thus, mature miRNA is not a reliable resource for studying Drosha processing events in some cases, and we believe that this might be the reason for the inconsistency between mature miRNA and 5′ moR results in several cases in the following experiments.

Altering the Upper Stem Length Also Causes Alternative Drosha Processing.

Next, we tested how the upper stem–ssRNA (terminal loop) junction regulates Drosha–DGCR8 complex action. The distance between the upper stem–ssRNA junction and the original Drosha cleavage site was reduced by deleting 1 or 2 bp of the upper stem at a position close to the upper stem–ssRNA junction in miR-150 and miR-122 (Fig. 2). Surprisingly, reducing the distance from the upper stem–ssRNA junction to the original Drosha cleavage site by deleting 1 or 2 bp also caused alternative Drosha processing (Fig. 2, Fig. S3, and Table S2). It appears that the upper stem–ssRNA junction contributes to the control of Drosha processing in a way that is similar, but not exactly the same, as the lower stem–ssRNA junction. Both data sets suggest that the Drosha–DGCR8 complex measures the distance from both the lower stem–ssRNA and upper stem–ssRNA junction to determine the Drosha cleavage site. Changing the distance by even 1 or 2 bp on either side of the cleavage site causes alternative Drosha processing.

Fig. 2.

Altering the distance from the upper stem–ssRNA (terminal loop) junction to the Drosha cleavage site can also cause alternative Drosha processing. (A) miR-150 D-C, with a 1-bp deletion in the upper stem close to the terminal loop. Deleted base pairs are indicated in gray. (B) miR-150 D-CA. (C) miR-122 D-U. (D) miR-122 D-UU.

Lower and Upper Stem–ssRNA Junctions Coordinate Microprocessor Processing.

WT pri-miR-150 and pri-mir-122 are mostly processed precisely by the Drosha–DGCR8 complex. Small distance changes from the lower stem–ssRNA junction or upper stem–ssRNA junction to the original Drosha cleavage site cause imprecise Drosha cleavage. We hypothesized that precise Drosha cleavage requires coordinated signals from both directions. The imprecision caused by an altered distance in one direction might be corrected by changing the distance from the other direction in compensation. To test this hypothesis, we changed the distance from both directions simultaneously by increasing the distance from the lower stem–ssRNA junction by 1 or 2 bp and reducing the distance from the upper stem–ssRNA junction by 1 or 2 bp at the same time to see whether it would shift Drosha cleavage precisely to another site from the original site.

As shown in Fig. 3A, altering the distance in both directions by 1 bp (miR-150 LS+A/D-C) caused significantly more cleavage at site 2 compared with changing the distance in only one direction (miR-150 LS+A or D-C; Figs. 1C, 2A, and 3A, Fig. S4A, and Table S2), suggesting that the cleavage shift toward site 2 could be enhanced by adding the two signals from both directions. When we inserted 2 bp in both directions at the same time (miR-150 LS+AU/D-CA), the Drosha cleavage site was precisely shifted to site 2 (Fig. 3B, Fig. S4B, and Table S2). For miR-122, altering by only 1 bp in both directions (miR-122 LS+U/D-U) is enough to shift the Drosha cleavage site 2 bp away from the original site 1 to site 2 exactly (Fig. 3C, Fig. S4C, and Table S2). Changing the distance in both directions by 2 bp (miR-122 LS+UG/D-UU) shifted the cleavage site mostly to site 2 but also a small fraction to site 3 (Fig. 3D, Fig. S4D, and Table S2). Thus, the imprecision caused by an altered distance in one direction could be rescued by adjusting the distance from the other direction in compensation, and we conclude that optimal distances in both directions are required for precision of the Drosha–DGCR8 complex processing.

Fig. 3.

The lower and upper stem–ssRNA junctions coordinate precise cleavage of the Drosha–DGCR8 complex. (A) miR-150 LS+A/D-C, with a simultaneous 1-bp insertion in the lower stem and a 1-bp deletion in the upper stem. (B) miR-150 LS+AU/D-CA. (C) miR-122 LS+U/D-U. (D) miR-122 LS+UG/D-UU.

Naturally Occurring Alternative Drosha Processing Can Be Reversed by Changing the Distances of the Lower and Upper Stem–ssRNA Junctions from the Cleavage Site.

miR-142 is one of the most abundant miRNAs in T cells (13). We have previously shown that the high frequency of 5′ isomiRs in miR-142 is caused by alternative Drosha processing (17). The pattern of naturally occurring alternative Drosha processing in miR-142 is similar to the pattern of alternative Drosha processing caused by reduced coordination between the lower and upper stem–ssRNA junctions by experimentally altering their distances from the Drosha cleavage site in miR-150 and miR-122. We hypothesized that naturally occurring alternative Drosha processing might also be caused by reduced coordination between the two signals.

To test this hypothesis, we made a series of mutations to the pri-miR-142 and checked the position of the Drosha processing site. We first checked the processing of WT pri-miR-142. Judging from the moR reads, Drosha cuts at three different sites (Fig. 4A and Table S2). Mature miR-142 reads also showed that Drosha indeed cuts at these three sites (Fig. S5A and Table S2). The data support our previous conclusion that the 5′ isomiR in miR-142 is caused by alternative Drosha processing. The pattern of miR-142 that we observed here in 293FT cells is consistent with our previously reported observation in naïve T cells, suggesting that the system we used here faithfully recapitulates the Drosha processing of miR-142 in naïve T cells (13, 17).

Fig. 4.

Naturally occurring alternative Drosha processing can be reversed by altering the lengths of the lower stem and upper stem. Illustration of Drosha cleavage sites for (A) WT miR-142 and (B and C) the indicated miR-142 mutations. The dotted line indicates the position of the original dominant Drosha cleavage site (site 2).

The majority of Drosha cleavages of the native pri-miR-142 were at site 2, which is 1 nt upstream of site 1 and 2 nt downstream of site 3 (Fig. 4A). To induce Drosha to precisely cut at site 1, we deleted 1 bp in the lower stem and/or inserted 1 bp in the upper stem (Fig. 4B). The 1-bp deletion in the lower stem (miR-142 LS-A) increased Drosha cleavage at site 1 from 35% to 75%, whereas the 1-bp insertion in the upper stem (miR-142 D+U) increased the cleavage to 77% (Fig. 4B and Table S2), supporting our previous observation that both the lower stem–ssRNA and the upper stem–ssRNA (terminal loop) junction regulate Drosha–DGCR8 complex action. The combination of both (miR-142 LS-A/D+U) induced Drosha to cut precisely at site 1 (97%; Fig. 4B and Table S2). The mature miRNA reads correlated well with the 5′ moR results—reads from site 1 were increased from 10% in WT miR-142 to 24%, 77%, and 91% in the three mutated miR-142s (Fig. S5B and Table S2). Thus, naturally occurring alternative Drosha processing can be reversed to cut precisely by altering the distance to the lower stem and upper stem junctions.

We also tried to induce Drosha to cut precisely at site 3 by inserting 2 bp in the lower stem and/or deleting 2 bp in the upper stem (Fig. 4C). Inserting 2 bp in the lower stem (miR-142 LS+AC) increased Drosha cleavage at site 3 from 1% to 75%, whereas deleting 2 bp in the upper stem (miR-142 D-UA) increased it to 41%, and the combination of both (miR-142 LS+AC/D-UA) induced Drosha to cut precisely at site 3 (97%; Fig. 4C and Table S2). Mature miRNA reads correlated well with 5′ moR results—reads from site 3 were increased from 35% in WT miR-142 to 88%, 76%, and 91% in the three mutated miR-142s (Fig. S5C and Table S2). The results appear to be similar to what we observed in constructs with a 1-bp deletion in the lower stem and/or a 1-bp insertion in the upper stem, but in the opposite direction. Thus, naturally occurring alternative Drosha processing can be reversed to cut precisely by altering the distance to the lower stem and upper stem junctions.

Discussion

In this study, we report two main findings. First, the Drosha–DGCR8 complex can sense signals from both the lower and upper stem determine the cleavage site, and coordinated signals from both are required for precise processing. Second, reduced coordination between the lower and upper stem–ssRNA junctions causes alternative Drosha processing, in which Drosha alternatively cleaves at different sites and generates multiple 5′ isomiRs, thus explaining how 5′ isomiRs are generated in some miRNAs.

Currently, there is no method available to reliably determine the Drosha processing site in human cells, which is one of the major obstacles to elucidating the mechanism of Drosha–DGCR8 recognition and processing of pri-miRNA. Our approach using moRs to determine the Drosha cleavage site provides a method to study Drosha–DGCR8 complex processing in human cells. The Drosha–DGCR8 complex plays a critical role not only in miRNA biogenesis but also in regulating mRNAs that have a hairpin structure (25–27). Unlike miRNAs, in which the mature molecule provides clues to where Drosha cleaves, the processing of mRNA substrates that do not generate mature miRNA is particularly difficult to study. Our approach provides a method to study Drosha–DGCR8 complex processing of these mRNA substrates, although the name moR is not accurate as there is no miRNA generated [Drosha cleavage–associated RNA (DcRNA) might be a better name]. However, because the mechanism of how moRs or DcRNAs are generated and preserved is not understood, exceptions may exist, in which the end of these RNAs might not faithfully represent the Drosha cleavage site.

How the Drosha–DGCR8 complex senses signals from both directions and why reduced coordination between the signals causes alternative Drosha processing are not clear. However, a recent study by Faller et al. showed that the highly cooperative binding of DGCR8 proteins to a pri-miRNA formed a higher-order structure that is big enough to cover the pri-miRNA, including the double-stranded region and surrounding ssRNA fragments, such as the basal segments and terminal loop (28). Thus, we propose that the DGCR8 complex could cover three helical turns of the pri-miRNA double-stranded region and also a small part of its surrounding ssRNA and therefore could sense signals from both the lower and upper stem to determine the Drosha cleavage site (Fig. S6). In miRNAs with optimal signals from both sides, such as in miR-150 and miR-122, the DGCR8 complex assembles to form a structure that allows Drosha to cleave precisely at one site, which ensures that only one mature miRNA is generated (Fig. S6A). In miRNAs, such as miR-142, the signals were evolutionarily selected not to be optimal for cleavage at a single site, so that the DGCR8 complex assembles into a structure that guides Drosha to cleave at multiple sites to generate multiple 5′ isomiRs (Fig. S6B), which could efficiently expand the pool of target genes.

Alternative Drosha processing was commonly observed in human cells when we changed the length of the lower/upper stem, which was not observed in previous reports that were mainly based on the in vitro systems (4, 8). Because additional regulatory factors other than DGCR8 and Drosha could affect the Drosha processing and each miRNA could be differentially associated with those regulatory factors (24, 29–32), the observed difference might have been caused by different choices of miRNAs. To examine this possibility, we generated serial mutations of miR-16, which has been comprehensively analyzed in previous reports (4, 8). Similar to miR-150 and miR-122, inserting 1 or 2 bp in the lower stem (miR-16 LS+A or LS+AG) caused alternative Drosha processing (Fig. 5 and Fig. S7). However, deleting 1 or 2 bp in the upper stem (miR-16 D-G or D-UG) did not change the Drosha processing site (Fig. 5 and Fig. S7), suggesting that the upper stem–ssRNA junction of miR-16 might not contribute to Drosha processing, which is consistent with previous reports (4, 8). However, altering the stem length by 1 bp in both directions (miR-16 LS+A/D-G) induces Drosha to cut more at an alternative site that is 1 bp upstream of the original site (from 7.6% to 27.5%) compared with inserting 1 bp in the lower stem only (miR-16 LS+A), and altering the stem length by 2 bp in both directions (miR-16 LS+AG/D-UG) induced Drosha to cut precisely at a site 2 bp upstream of the original site compared with inserting 2 bp in the lower stem only (miR-16 LS+AG, with only 26.5% cleavage at the site; Fig. 5 and Fig. S7), suggesting that the upper stem of miR-16 can contribute to the selection of the Drosha processing site and the lower and upper stem–ssRNA junctions coordinate selection of the Drosha processing site. These data also suggested that, whereas optimal distances on both sides are generally required for precise Drosha processing, alteration of the distance on one side might be tolerated in some miRNAs, such as miR-16. Thus, although the model we proposed might have broad generality, it is possible that there might be exceptions.

Fig. 5.

Validating the model in miR-16. Drosha cleavage sites for the indicated miR-16 mutations. Representative reads of cloned small RNAs are found in Fig. S7A.

Our results concerning Drosha–DGCR8 complex recognition and processing of pri-miRNA could provide clues for designing better miRNA-based shRNA. It is clear that both distances (from the lower stem and upper stem–ssRNA junction to the Drosha cleavage site) should be optimally maintained to ensure precise processing of shRNA so that the desired siRNA duplex is generated. When the potency of shRNA is in doubt, it is also possible to manipulate the distance between the lower and upper stem–ssRNA junctions to purposefully make Drosha cut at different sites so that multiple siRNA duplexes are generated, which could double or triple the chance to obtain a high-potency shRNA, as even a 1-bp change at the end of the siRNA duplex can dramatically alter siRNA functionality. Additional discussion is available in SI Discussion.

Materials and Methods

Small RNAs were cloned partly as described previously (13, 33). Here we made major modifications to improve adaptor ligation efficiency and thereby minimize cloning bias. Briefly, the small RNAs were purified with the miRNeasy kit (Qiagen). Small RNA (50 ng) was ligated with 3′ and 5′ linkers (with barcode) with an improved ligation method. The ligated small RNAs were reverse-transcribed and amplified with the KAPA library amplification kit (KAPA Biosystems) for 10 cycles, and the library was sequenced using the Illumina HiSeq2000 or MiSeq. The deep sequencing data were processed by in-house–developed software. All reads that had been cloned only once were discarded to lower the noise level. All reads of cloned small RNAs for indicated miRNAs are included in Dataset S5. Note that for small RNAs to be efficiently cloned with the method described here, the 5′ phosphate and 3′ hydroxyl groups must be preserved. Additional methods are available in SI Materials and Methods.