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• W1985974546 abstract "Article25 November 2004free access Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha Yan Zeng Yan Zeng Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Rui Yi Rui Yi Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USAPresent address: Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA Search for more papers by this author Bryan R Cullen Corresponding Author Bryan R Cullen Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Yan Zeng Yan Zeng Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Rui Yi Rui Yi Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USAPresent address: Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA Search for more papers by this author Bryan R Cullen Corresponding Author Bryan R Cullen Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Author Information Yan Zeng1, Rui Yi2 and Bryan R Cullen 1,2 1Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA *Corresponding author. Duke University Medical Center, Box 3025, Durham, NC 27710, USA. Tel.: +1 919 684 3369; Fax: +1 919 681 8979; E-mail: [email protected] The EMBO Journal (2005)24:138-148https://doi.org/10.1038/sj.emboj.7600491 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A critical step during human microRNA maturation is the processing of the primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate the ∼60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA substrates and selects its cleavage sites has remained a mystery, especially given that the known targets for Drosha processing show no discernable sequence homology. Here, we show that human Drosha selectively cleaves RNA hairpins bearing a large (⩾10 nucleotides) terminal loop. From the junction of the loop and the adjacent stem, Drosha then cleaves approximately two helical RNA turns into the stem to produce the precursor microRNA. Beyond the precursor microRNA cleavage sites, approximately one helix turn of stem extension is also essential for efficient processing. While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen. Introduction First discovered in Caenorhabditis elegans, microRNAs (miRNAs) are a large family of ∼22-nucleotide (nt)-long RNAs widely expressed in metazoan eukaryotes (Lee et al, 1993, 2004; Ruvkun et al, 2004). An estimated 1% of animal genes encode miRNAs (Bartel, 2004). While the functions of miRNAs are only beginning to be appreciated, it is generally believed that miRNAs regulate gene expression at the post-transcriptional level by inhibiting the expression of mRNAs bearing fully or partly homologous target sequences (Carrington and Ambros, 2003; Bartel, 2004; He and Hannon, 2004; Novina and Sharp, 2004). Like most other cellular RNAs, miRNAs undergo a maturation process (Murchison and Hannon, 2004). miRNAs are initially transcribed as part of a long primary miRNA (pri-miRNA) transcript, which contains the mature miRNA as part of a predicted RNA hairpin. In animal cells, the first notable step in miRNA processing occurs when Drosha, a nuclear RNaseIII enzyme, excises the upper part of this RNA hairpin to generate the precursor miRNA (pre-miRNA), which is ∼60 nt long with a 3′ 2 nt overhang (Lee et al, 2002, 2003). The pre-miRNA is then exported to the cytoplasm by the nuclear export factor Exportin 5 and the Ran-GTP cofactor (Yi et al, 2003; Bohnsack et al, 2004; Lund et al, 2004). The second cleavage step takes place in the cytoplasm where Dicer, another RNaseIII enzyme, cuts near the hairpin loop to release an ∼22-base-pair (bp) imperfect RNA duplex intermediate with ∼2 nt 3′ overhangs at both ends (Grishok et al, 2001; Hutvágner et al, 2001; Ketting et al, 2001). Usually, only one of the two RNA strands is stable in vivo. This polarity arises from the fact that the RNA-induced silencing complex (RISC), or a related complex, identifies the strand within the duplex with weaker hydrogen bonding at its 5′ end and then selectively incorporates this strand into RISC (Khvorova et al, 2003; Schwarz et al, 2003). The opposite strand, denoted as miRNA*, is released by RISC and generally rapidly degraded. However, in the rare cases where hydrogen bonding at the two ends of the miRNA duplex intermediate is equivalent, either strand may be randomly incorporated into RISC. The expression of several hundred miRNAs has been experimentally verified in numerous organisms and cells, and computer programs have been designed to predict more miRNAs on a genome-wide basis (Bartel, 2004). Although mature miRNAs are all ∼22 nt in size, and the secondary structures of pre-miRNAs are broadly similar, the sequences of both miRNAs and pre-miRNAs are very diverse, and the predicted pre-miRNA secondary structures can be quite different in detail. For example, the stem regions may contain different numbers of unpaired residues at different locations, and computer-predicted terminal loops range from 3 nt to well over 10 nt in size (Lagos-Quintana et al, 2001; Lau et al, 2001; Lee and Ambros, 2001). This raises the obvious question of how these unrelated RNA hairpins can be recognized by the same processing enzymes and then precisely processed to yield mature miRNAs. Drosha has emerged as a key determinant of which part of the pri-miRNA will become the mature miRNA. As mentioned above, Drosha cleaves pri-miRNAs to yield pre-miRNAs and thereby generates one end of the mature miRNA. Dicer recognizes the 3′ 2 nt overhang of pre-miRNAs and then cuts ∼22 nt away to produce the miRNA:miRNA* duplex (Zhang et al, 2002, 2004). miRNAs are then selected over miRNAs* by RISC according to the 5′ end base-pairing rule (Khvorova et al, 2003; Schwarz et al, 2003). Therefore, Drosha plays a critical role in deciding the sequence and abundance of miRNAs, as the initial cleavage sites chosen by Drosha largely dictate where Dicer will cleave and, hence, which miRNA strand enters RISC (Bartel, 2004). In this study, we have analyzed miRNA processing and expression in human cells transfected with plasmids encoding wild-type and mutant pri-miRNAs, and have complemented these in vivo experiments with in vitro Drosha processing assays. Our results indicate that, within the context of pri-miRNAs, RNA stem–loops with a large, unstructured terminal loop (⩾10 nt) are the preferred substrates for Drosha cleavage, and that Drosha then cleaves ∼22 nt away from the loop/stem junction. A continuation of the pri-miRNA stem, outside of the mature pre-miRNA, is also critical for miRNA processing and can slightly modify the precise cleavage sites used for pre-miRNA production. Results Pri-miR-30a processing requires a large terminal loop We have previously used the CMV immediate-early promoter to overexpress pri-, pre-, and mature miR-30a in transfected human cells, as a means to study miRNA biogenesis (Zeng et al, 2002; Zeng and Cullen, 2003). The construct used, here termed pCMV-miR-30a, expresses the 73 nt miR-30a sequence shown in Figure 1A. This plasmid gives rise to readily detectable levels of two mature miRNAs, termed miR-30a-5p and miR-30a-3p, in transfected cells (miR-30a is unusual in giving rise to two mature miRNAs). By mutagenesis, we have previously identified two features of the pri-miR-30a hairpin that are important for miRNA expression (Zeng and Cullen, 2003). One is base pairing near the base of the stem, beyond the pre-miRNA sequence, as the pCMV-miR-30a(GAG) mutant (Figure 1A) makes no detectable miRNAs when transfected into 293T cells. The other is the terminal loop. Some RNA folding programs (e.g., MFOLD) predict that the apex of the pre-miR-30a hairpin folds to form a 5 nt bulge, a 3 bp stem, and finally a 4 nt terminal hairpin loop (Figure 1A), yet we had found that disruption of the predicted 3 bp stem had no effect, while deleting the 5 nt bulge, thereby extending the predicted stem by 3 bp and leaving the 4 nt loop intact, severely reduced miRNA expression (Zeng and Cullen, 2003). We therefore considered the possibility that pre-miR-30a might instead contain an unstructured 15 nt terminal loop. Figure 1.Analysis of pri-miR-30a hairpin terminal loop mutants. (A) Computer-predicted secondary structure of the pri-miR-30a hairpin (Lagos-Quintana et al, 2001) and sequences of the miR-30a terminal loop mutants. The mutation introduced into miR-30a(GAG), a miR-30a null mutant, is indicated. The boxed sequence derives from appended restriction sites and facilitates miR-30a expression. The arrowheads identify the major cleavage sites for endogenous pre-miR-30a. (B) Northern analyses of miR-30a-5p (upper panel) and miR-30a-3p (lower panel) expression in 293T cells transfected with the indicated pri-miR-30a expression plasmids (lanes 3–10). Lane 1; DNA size markers; lane 2; RNA from nontransfected 293T cells. 5S rRNA was used as a loading control. (C) A reporter assay to compare miR-30a-3p function when expressed from pCMV- or pSuper-based vectors. The firefly luciferase-based reporter, pCMV-luc-8xmiR-30a(P), was cotransfected into 293T cells along with pRL-CMV (Promega), an internal control Renilla luciferase expression plasmid, and pCMV-miR-30a or pSuper-miR-30a variants. The ratio of firefly luciferase activity relative to Renilla luciferase activity from cells transfected with an empty pCMV or pSuper vector (−) is set at 1.00. Average of three experiments with standard deviation is shown. (D) Pre-miR-30a and mature miR-30a-3p expression in 293T cells transfected with the pSuper-miR-30a plasmids was determined by Northern blotting, as shown in panel B. miRNA expression levels induced by pSuper-miR-30a and pCMV-miR-30a are comparable (data not shown). Download figure Download PowerPoint To examine the contribution of terminal loop size and sequence in more detail, pCMV-miR-30a mutants (L5–L15) were constructed with loop sizes ranging from 5 to 15 nt. The terminal loop sequences used either represented deletions of the natural pre-miR-30a terminal loop (L12 and L9.1) or random terminal loop sequences (L5, L7, L8, L9.2, and L15) (Figure 1A). Northern analyses showed that L5, L7, and L8 made very little mature miRNA (Figure 1B, lanes 4–6). In contrast, L9 (both L9.1 and L9.2; Figure 1B, lanes 7 and 8) had improved miRNA production, while L12 and L15 were essentially wild type (Figure 1B, lanes 9 and 10). Variants of L7, L8, and L9 with different loop sequences gave similar results (Figure 1B and data not shown), thus demonstrating that the size of the loop is more important than the sequence per se, although this of course ultimately dictates the loop structure. In all the mutants that were defective in mature miRNA production, pre-miRNA levels were also diminished (Figure 1B). Primer extension experiments confirmed the Northern blotting results (data not shown). Moreover, experiments designed to measure the biological activity of miR-30a in transfected cells, using a previously described indicator construct that contains eight fully complementary target sites for miR-30a-3p linked to the firefly luciferase gene (Zeng et al, 2003), revealed that the L9.2, L7, and L5 mutants of pCMV-miR-30a were increasingly attenuated in their ability to inhibit luciferase gene expression (Figure 1C). Production of a mature, stable miRNA from the initial pri-miRNA transcript requires nuclear processing by Drosha, nuclear export by Exportin 5, cytoplasmic processing by Dicer, and finally incorporation of the mature miRNA into RISC. While any of these steps could be affected by terminal loop size, we favored the hypothesis that the first step, that is, Drosha processing, was primarily affected in mutants of pri-miR-30a bearing small terminal loops. If this is indeed the case, then direct production, in the nucleus, of a transcript identical to the ∼63 nt pre-miR-30a intermediate, using RNA polymerase III (polIII), should rescue both mature miR-30a production and function. In fact, expression of the L5, L7, and L9.2 mutants as a pre-miR-30a transcript entirely (L7 and L9.2) or largely (L5) rescued both mature miR-30a-3p function (Figure 1C) and expression (Figure 1D) in transfected cells. These data, together with data showing that recombinant Dicer is able to process wild-type pre-miR-30a, as well as the L5 and L9.2 mutants, into mature miR-30a with equivalent efficiency in vitro (not shown) argue that terminal loop deletion mutations are inhibiting a step prior to nuclear export of the pre-miRNA, that is, most likely Drosha processing of the pri-miRNA. Efficient processing of other pri-miRNAs also requires a large terminal loop Our results with miR-30a indicated that a terminal loop of ⩾10 nt in size is an important determinant of efficient pri-miR-30a processing in vivo (Figure 1). If this is a general property, then other pre-miRNAs would also be predicted to contain terminal loops of ⩾10 nt. However, computer predictions of the structure of other miRNA precursors frequently predict terminal loops that are much smaller. To determine if a large terminal loop is a general feature of pri-miRNAs, we next tested a second human miRNA termed miR-21. Computer folding programs predict that pre-miR-21 has a 5 nt terminal loop adjacent to a 4 bp stem containing two G:U base pairs (Lagos-Quintana et al, 2001; Figure 2A). Mutations were introduced to either enlarge the loop or to stabilize this stem in the context of the pri-miR-21 expression plasmid pCMV-miR-21 (Zeng and Cullen, 2003), and the resultant plasmids were transfected into cells. To detect functional miR-21, we employed a reporter assay and also performed primer extension and Northern blotting (Figure 2B). We have previously described a reporter construct that encodes the firefly luciferase gene linked to eight copies of a target sequence perfectly complementary to miR-21 and that is markedly downregulated in response to miR-21 overexpression (Zeng et al, 2003). Both the reporter assay and RNA analyses showed that while opening up the loop had no effect on mature miR-21 expression, stabilizing the predicted stem, therefore restricting the loop size, was deleterious (Figure 2B). Thus, the single nucleotide mutations M2 and M4, which are predicted to destabilize the 4 bp stem shown in Figure 2A, had no effect on either miR-21 function or expression (Figure 2B, lanes 5 and 7), while the point mutations M1 and, particularly, M5, which are predicted to stabilize the 4 bp stem, attenuated both miR-21 function and expression (Figure 2B, lanes 4 and 8). Moreover, a mutation predicted to strongly stabilize the 4 bp stem, termed miR-21(CCG), essentially abolished miR-21 production and function (Figure 2B, lane 3). These data suggest that the miR-21 terminal loop as predicted in silico (Figure 2A) is smaller than actually found in vivo, or that the loop structure is dynamic in vivo, with cellular processing factors perhaps stabilizing or inducing a larger loop (Figure 3). Figure 2.Analysis of pri-miR-21 loop mutants. (A) Schematic of the computer-predicted miR-21 secondary structure, point mutations (1, 2, 3, 4, and 5), and the miR-21(GGU) and miR-21(CCG) mutants. Mature miR-21 is underlined, and the arrowheads indicate the cleavage sites used to produce the predicted pre-miR-21. (B) Analyses of miR-21 loop mutants. Luciferase assays were performed as in Figure 1C and the similar indicator construct pCMV-luc-8xmiR-21(P) was used as the reporter. Primer extension and Northern blotting assays were performed using oligonucleotide probes described in Supplementary Table 1. 5S rRNA was used as a loading control in the Northern analysis. Download figure Download PowerPoint Figure 3.Alternative structures for miR-30a, miR-21, miR-27a, and miR-31. Comparison of the published, computer-predicted secondary structures (Lagos-Quintana et al, 2001) with possible new secondary structures proposing larger terminal loops for these miRNA precursors. These proposed structures are based on the mutational analyses, and transfections and in vitro assays, performed in this paper. These larger loops may form as indicated or may be opened by Drosha binding. Mature miRNA sequences are underlined, and predicted or confirmed Drosha cleavage sites are shown by arrows. Download figure Download PowerPoint Two more human miRNAs were also analyzed for the effect of terminal loop size on Drosha processing efficiency. Genomic DNA (∼250 bp), centered on the predicted ∼80 nt pri-miR-27a and pri-miR-31 RNA hairpins, was cloned into the polIII-based expression plasmid pSuper (Brummelkamp et al, 2002). These plasmids are therefore predicted to express pri-miRNAs transcribed by polIII that are analogous to the polII-transcribed pri-miRNAs analyzed in Figures 1 and 2. As shown in Figure 4A, pre-miR-27a is predicted by computer analysis to fold into an RNA hairpin bearing an 8 nt terminal loop flanked by a single base pair, a 1 nt bulge, a 2 bp stem, and opposing 1 nt bulges. However, pre-miR-27a could instead form a 17 nt terminal loop above a 7 bp stem (Figure 3). In the case of pre-miR-31, the computer predicts an 8 nt terminal loop flanked by a 3 bp stem and three bulged nucleotides (Figure 4B), but we considered that pre-miR-31 might instead contain an unstructured 17 nt terminal loop (Figure 3). Mutations that are predicted to affect the size of the terminal loop were then introduced into the miR-27a and miR-31 expression plasmids, as shown for miR-21 in Figure 2A. Northern blotting showed that the production of both pre-miRNAs and mature miRNAs was reduced when the short stems predicted to be adjacent to either 8 nt terminal loop were stabilized or extended into the loops for both miR-27a (Figure 4A, mutants 1 and 2) and miR-31 (Figure 4B, mutants 1 and 3). In contrast, a mutation designed to destabilize the 3 bp stem predicted to be adjacent to the 8 nt terminal loop in pre-miR-31 had no effect (Figure 4B, lane 3). These data, while limited, are therefore entirely consistent with the mutational analysis of pre-miR-30a and pre-miR-21 (Figures 1 and 2) and suggest that a large (⩾10 nt) terminal loop may be a general requirement for efficient pri-miRNA processing. Figure 4.Analysis of loop mutants of human pri-miR-27a and pri-miR-31. (A) miR-27a expression. Plasmids encoding wild-type pri-miR-27a, or one of two mutants (1 and 2), were cotransfected with pH1-GFP into 293T cells. Northern blotting was performed to detect the expression of pre- and mature miR-27a (upper panel) and the control GFP siRNA (lower panel). (B) miR-31 expression. Three pri-miR-31 mutants were constructed (boxed sequences), and their miRNA expression levels compared to wild-type miR-31 in transfected 293T cells. Download figure Download PowerPoint Drosha requires a large terminal loop for pri-miRNA processing in vitro To test directly if Drosha discriminates against pri-miRNA substrates bearing small loops, we next performed in vitro processing assays using FLAG-tagged Drosha enzyme that had been isolated from overexpressing 293T cells by immunoaffinity purification. It is important to note that this Drosha preparation is likely to contain other cellular factors, including any proteins that remain bound to Drosha during the purification process. Incubation of a 32P-labeled pri-miR-30a substrate RNA with FLAG immunoprecipitates obtained from FLAG-tagged Drosha overexpressing 293T cells, or from control 293T cells, yielded several RNA cleavage products that were only observed, or were much stronger, in the former case (Figure 5). The position of pre-miR-30a (‘*’) was inferred by its size (63 nt) and by its complete absence in the miR-30a(GAG) lane. The miR-30a(GAG) (Figure 1A) mutant is incapable of giving rise to detectable levels of either the pre-miRNA intermediate or the mature miRNA in transfected cells (Zeng and Cullen, 2003) and a similar pri-miR-30a mutant was previously shown to be resistant to in vitro processing by whole-cell lysates (Lee et al, 2003). This mutant therefore serves as a negative control. As shown in Figure 5, there was a good correlation between the expression of pre-miR-30a in transfected cells (Figure 1) and the ability of Drosha to produce pre-miRNA in vitro. Notably, the L5 and L9.2 mutants of miR-30a, which give rise, respectively, to almost undetectable or reduced levels of mature miR-30a in transfected cells (Figure 1B) also gave rise to essentially undetectable or reduced levels of pre-miR-30a in vitro (Figure 5). We consistently observed an additional RNA band (indicated by an arrowhead at the right of Figure 5) that ran slightly higher than the pre-miR-30a marked by ‘*’. The identity of this upper band is not known, but it is not the final pre-miR-30a product, as RNA isolated from the band is not a substrate for Dicer, while RNA from the lower band is (Figure 7B). It is possible that this band is derived from one of the two flanking sequences that are predicted also to be produced by Drosha cleavage of the pri-miR-30a RNA probe used, and that are expected to be ∼78 and ∼61 nt in length. Figure 5.In vitro Drosha cleavage of pri-miR-30a transcripts. FLAG immunoprecipitates from mock-transfected 293T cells (−) or cells transfected with pCK-Drosha-FLAG(+) were incubated with a 32P-labeled ∼202 nt RNA probe encoding the indicated miR-30a variants. RNA cleavage products were recovered and resolved on a denaturing 10% polyacrylamide gel. The pre-miRNA band is indicated by an asterisk, and the background band running slightly above by an arrowhead. Cleavage efficiency was calculated as the intensity of the pre-miRNA band divided by that of the remaining substrate, and set as 100% for the wild-type transcript. DNA size standards are shown next to the autoradiograph. Download figure Download PowerPoint Figure 6.Analysis of the effects of changes in pri-miR-30a stem length. (A) Predicted hairpin RNA structures adopted by pri-miR-30a variants with different stem extensions beyond the pre-miR-30a structure. Boxed sequences represent added restriction sites. The positions of the 5′ and 3′ ends of the endogenous pre-miR-30a are indicated by arrows in E17. (B) Primer extension experiments to determine the expression level and 5′ ends of mature miR-30a-3p (left panel) or miR-30a-5p (right panel) variants. The sequences of the miR-30a-3p std1 and miR-30a-5p std1 and 2 size standards are given in Supplementary Table 1. (C) The 5′ cleavage sites mapped in panel B are indicated as I, II, III, and IV on the predicted pri-miR-30a RNA structure. Two potential duplex intermediates formed after cleavage are indicated (3′ ends are inferred). (D) In vitro Drosha processing of transcripts encoding E10 and E21. The asterisk indicates the pre-miRNA band. The position of a 66 nt DNA size marker is indicated. Download figure Download PowerPoint To further confirm the hypothesis that Drosha preferentially cleaves pri-miRNAs bearing a large (⩾10 nt) terminal loop, we performed additional in vitro cleavage assays using RNA probes derived from wild-type and selected mutant forms of the pri-miR-21 (Supplementary Figure 1A) or pri-miR-31 (Supplementary Figure 1B) RNAs. These data demonstrated that pri-miR-21 (Figure 2) and pri-miR-31 (Figure 4B) terminal loop mutants that inhibit mature miRNA production in transfected cells also inhibit Drosha cleavage of the pri-miRNA precursor in vitro. Role of the pri-miRNA stem extension during miRNA expression As noted above, in addition to an optimal terminal loop, a continuation of base pairing beyond the base of the pre-miRNA stem is also critical for miRNA processing, as pCMV-miR-30a(GAG) (Figure 1A) and pCMV-miR-21(GGU) (Figure 2A) made no pre-miRNAs or mature miRNAs in transfected cells (Zeng and Cullen, 2003), and both also failed to give rise to pre-miRNAs in Drosha cleavage assays in vitro (Figure 5 and Supplementary Figure 1A). To test how long a stem extension is required, and how it might affect pri-miRNA processing, more miR-30a variants (Figure 6A) were made. These mutants were named according to the distance from the first predicted base pair outside the pre-miRNA to the 5′ end of the endogenous pre-miR-30a intermediate (position shown in E17). These extensions are predicted to be mostly double stranded but also include small bulges (Figure 6A). Thus, the E17 mutant contains an extension beyond the endogenous pre-miR-30a intermediate that is predicted to add 14 bp, and several unpaired nucleotides, to the base of the pre-miR-30a RNA hairpin (Figure 6A). Pri-miRNA sequences located outside the structures shown were predicted to be largely single stranded. Figure 7.Movement of the loop/stem junction affects Drosha cleavage site selection. (A) Schematic of the predicted RNA secondary structures adopted by the J+1 and J−1 mutants of pCMV-miR-30a(E10). Mutated residues are underlined and the arrows indicate the 5′ ends of the miRNAs, as defined in the primer extension shown in the lower panel. Sequences of the size standards used are given in Supplementary Table 1. (B) Drosha processing of miR-30a transcripts in vitro. M: DNA markers. Pre-miRNA bands are indicated by asterisks. (C) To test the identities of Drosha cleavage products, RNAs from bands A, B, and C in panel B were isolated, digested with recombinant human Dicer, and resolved on a 15% denaturing gel. Download figure Download PowerPoint Plasmids encoding these pri-miR-30a variants, driven by the CMV promoter (Zeng et al, 2002), were transfected into 293T cells, and primer extension experiments were performed to determine the expression levels and 5′ ends of overproduced miR-30a-5p and miR-30a-3p. As shown in Figure 6B, E5 produced few mature miRNAs. E10 (which is similar to wild-type pri-miR-30a) and E12 gave expression patterns that were similar, in terms of both expression level and the 5′ ends of the observed miRNAs. In contrast, E17 yielded mostly miR-30a-5p. The last variant tested, E21, was largely defective in producing any miRNAs, indicating that too long a stem extension is also undesirable. Northern analyses confirmed the expression patterns of these variants, and pre-miRNA levels correlated with mature miRNA levels (data not shown). Based on published reports (Lee et al, 2003; Zeng and Cullen, 2003) and the data presented here, we therefore conclude that a modest, ⩾8 bp stem extension, beyond the 5′ end of a pre-miRNA, is required for optimal miRNA processing from a long pri-miRNA. The primer extension analysis (Figure 6B) showed that the three pri-miR-30a stem mutants able to produce mature miRNA effectively, that is, E10, E12, and E17, had minor but distinct differences in their processing sites. Specifically, both E10 and E12 actually produced both forms of miR-30a, while E17 produced very little miR-30a-3p (Figure 6B). The two miR-30a RNAs produced by E10 and E12 appear to be almost equal mixtures of miRNAs that differ by 1 nt, with the longer form being full-length miR-30a-3p (cleavage site III; Figure 6C) while the second form is 1 nt shorter (cleavage site IV; Figure 6C). The limited level of miR-30a-3p produced by E17 appears to be of the shorter form. Analysis of the miR-30a-5p strand by primer extension again showed two products, differing by 1 nt, for E10. E12 appeared to produce primarily the shorter form, while E17 produced only the longer (Figure 6B). Based on the size standards used, E10 appears to be cleaved at sites I and II (Figures 6C and 7A), while E12 is predominantly cleaved at site II and E17 at site I (Figure 6C). Based on this analysis, it is possible that E10 could give rise to four different duplex intermediates, E12 to two, and E17 to one. The duplex intermediate predicted for E17 is shown as duplex B in Figure 6C. As may be observed, this duplex contains an A:U base pair at one end and a G:C base pair at the other, thus strongly favoring the incorporation into RISC of the miR-30a-5p strand, whose 5′ end forms part of an A:U base pair, over the miR-30a-3p strand, whose 5′ end is predicted to form part of a more stable G:C base pair (Schwarz et al, 2003). In contrast, duplex A, which is predicted to be produced by processing of both the E10 and E12 pri-miRNAs, should form G:C base pairs at both ends, thus predicting similar levels of incorporation of miR-30a-3p and miR-30a-5p into RISC, and similar stability, as is indeed observed (Figure 6B). As the E17 mutation moves the cleavage site chosen by Drosha 1 nt away from the terminal loop, does this imply that longer stem lengths invariably have this effect? The answer to this question is clearly no, as the E21 mutant, bearing the longest stem extension tested, actually produced a low but det" @default.
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• W1985974546 title "Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha" @default.
• W1985974546 cites W144423133 @default.
• W1985974546 cites W1967633197 @default.
• W1985974546 cites W1974227032 @default.
• W1985974546 cites W1980269624 @default.
• W1985974546 cites W1984109071 @default.
• W1985974546 cites W1984403668 @default.
• W1985974546 cites W1992941735 @default.
• W1985974546 cites W2004812096 @default.
• W1985974546 cites W2014064723 @default.
• W1985974546 cites W2020458511 @default.
• W1985974546 cites W2022646895 @default.
• W1985974546 cites W2027689967 @default.
• W1985974546 cites W2038783205 @default.
• W1985974546 cites W2049392988 @default.
• W1985974546 cites W2054109341 @default.
• W1985974546 cites W2057906056 @default.
• W1985974546 cites W2059382687 @default.
• W1985974546 cites W2071596792 @default.
• W1985974546 cites W2082213106 @default.
• W1985974546 cites W2103392842 @default.
• W1985974546 cites W2111436354 @default.
• W1985974546 cites W2117391818 @default.
• W1985974546 cites W2119015197 @default.
• W1985974546 cites W2122575917 @default.
• W1985974546 cites W2136148915 @default.
• W1985974546 cites W2136991248 @default.
• W1985974546 cites W2138664009 @default.
• W1985974546 cites W2146642335 @default.
• W1985974546 cites W2149291473 @default.
• W1985974546 cites W2151922790 @default.
• W1985974546 cites W2152869747 @default.
• W1985974546 cites W2154841861 @default.
• W1985974546 cites W2157569278 @default.
• W1985974546 cites W2165804743 @default.
• W1985974546 cites W2167557255 @default.
• W1985974546 cites W4249555503 @default.
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