Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis

research paper RNA Biology 8:6, 1105-1114; November/December 2011; © 2011 Landes Bioscience

Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis Steven G. Chaulk,1,* Gina L. Thede,1 Oliver A. Kent,2 Zhizhong Xu,1 Emily M. Gesner,1 Richard A. Veldhoen,1 Suneil K. Khanna,1 Ing Swie Goping,1 Andrew M. MacMillan,1 Joshua T. Mendell,3 Howard S. Young,1,4,* Richard P. Fahlman1,5,* and J.N. Mark Glover1,* Department of Biochemistry; 4National Institute for Nanotechnology; 5Department of Oncology; University of Alberta; Edmonton, AB Canada; 2 Johns Hopkins University Institute of Genetic Medicine; Baltimore, MD; 3Department of Molecular Biology; University of Texas Southwestern Medical Center; Dallas, TX USA

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Key words: pri-miRNA structure, miRNA cluster, drosha, miRNA biogenesis

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumorigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction

Small RNA species have been found to regulate gene expression in an increasing number of biological processes.1-4 One class of small RNAs, miRNAs (microRNAs), are initially expressed in much larger primary miRNAs (pri-miRNA) that contain one or several miRNA sequences that are located within predicted ~70 nucleotide stem-loop (hairpin) structures1,5-8 (Figs. 1A and S1). The first processing step in miRNA biogenesis relies on the RNase III enzyme Drosha to release the ~70 nucleotide miRNA containing hairpins from the pri-miRNA. These precursor miRNAs (premiRNA) are then exported out of the nucleus to the cytoplasm by Exportin 5/Ran GTPase and are processed by another RNase III, Dicer, into mature ~22 nucleotide miRNAs.8,9 The miRNAs are subsequently incorporated into RISC (RNA-induced silencing complex) which directs RNAi mediated gene regulation by targeting a complementary mRNA.10 Several miRNA genes have been identified that function as oncogenes (termed OncomiRs) or tumor suppressors.4,6,11,12 One OncomiR is miR-17~92 (OncomiR-1), which is located at 13q31.3, a genomic locus that is amplified in several types of lymphomas and other human cancer types.13-15 Expression of miR-17~92 can have profound effects on tumorigenesis and development in mouse model systems. Overexpression of miR-17~92

in a mouse B cell lymphoma model enhances disease progression and morbidity. In contrast, knock-out of miR-17~92 along with one or both of its paralogues (miR-106b and miR-106a~363) results in death at mid-gestation.3 Regulation of gene expression by miRNAs has emerged as a very complex process. An intriguing but unaddressed question is why miRNAs are often expressed as clusters within a single transcript.16,17 While several studies have shown specific secondary structure requirements for the processing of these miRNAs by Drosha,18-20 as well as a role for the miRNA hairpin looped nucleotides in mRNA targeting,21,22 the possibility of a role for higher order structure in these pri-miRNA clusters has not been addressed. Here we report that the miR-17~92 miRNA cluster adopts a compact globular structure where the 5' region of the cluster folds on a 3' core domain that contains the miR-19b and miR-92 hairpins. The internalized miRNAs are processed less efficiently than those on the surface of the structure. Disruption of the tertiary structure of the cluster exposes the buried miRNAs enabling more efficient processing and miRNA maturation. The increased processing of miR-92 results in an increased repression of a validated miR-92 target, ITGA5 mRNA. Our findings establish a role for RNA tertiary structure in miRNA biogenesis which in turn impacts the regulatory activity of the individual miRNAs.

*Correspondence to: Steven G. Chaulk, Howard S. Young, Richard P. Fahlman and J.N. Mark Glover; Email: [email protected], [email protected], rfahlman@ ualberta.ca and [email protected] Submitted: 05/26/11; Revised: 07/25/11; Accepted: 07/28/11 DOI: 10.4161/rna.8.6.17410 www.landesbioscience.com

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©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 1. miR-17~92 processing. (A) Primary sequence organization of the miRNA hairpins of pri-miR-17~92 and its processing by Drosha to premiRNAs and by Dicer to the mature miRNAs. (B) Time course of Drosha (Microprocessor) processing of miR-17~92 to yield pre-miR-17, pre-miR-18, premiR-19a, pre-miR-20, pre-miR-19b and pre-miR-92. Northern blotting using probes targeting the mature miRNA sequence (terminal loop sequences for miR19a and miR19b were targeted) detect both the pri- and pre-miRNA. Positions of pri-miRNA and pre-miRNA products are indicated. (C) Plots of fraction of pre-miRNA product relative to pri-miRNA precursors versus time using data averaged from two independent experiments give kobs values of: miR-17 0.52 ± 0.18; miR-18 0.027 ± 0.019; miR-19a 0.68 ± 0.03; miR-20 0.43 ± 0.03; miR-19b 0.11 ± 0.06; miR-92 0.03 ± 0.02. (D) Denaturing PAGE analysis of RNAse T1 digestion of c13orf25 intron 3. The *indicates RNA sizes of 2–10.0 kb. Undigested 3.7 kb intron 3 ((-) lane), ~800 and ~600 nucleotide RNase T1 resistant fragments of intron 3 ((+) lane) resolved by denaturing PAGE. The ~800 nucleotide product contains miR-17~92. (E) Native gel electrophoresis reveals a magnesium-dependent change in electrophoretic mobility of miR-17~92. Left part, miR-17~92 annealed and electrophoresed in the absence of magnesium has a mobility similar to an ~800 base pair DNA. Right part, miR-17~92 annealed and electrophoresed in the presence of magnesium has a similar mobility to a 400 base pair DNA marker.

Results Drosha processing of miR-17~92. We were intrigued by the polycistronic character of the miR-17~92 miRNA cluster.23,24 Approximately 50% of human miRNAs are organized into clusters, the majority of which (~60%) have a maximum intermiRNA distance (MID) of 1 kb or less.16,17 miR-17~92 is a miRNA cluster encoding six miRNAs with a MID of 0.8 kb where only ~50% of the bases are within miRNA hairpin structures (Figs. 1 and S1). The simultaneous expression of multiple miRNAs within a single primary transcript (Fig. 1A) provides an opportunity to coordinate the expression levels of the mature miRNAs. In vitro Drosha processing reactions25-27 using immunoprecipitated Microprocessor complex (overexpressed Drosha and DGCR8 in HEK293T cells) and in vitro transcribed miR-17~92

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cluster, under physiological Mg2+ concentration,28 revealed different efficiencies of Drosha processing (Fig. 1B and C) with the generation of the pre-miR-17, pre-miR-19a and pre-miR-20 hairpins being much more efficient (up to ~17 fold for miR-17 versus miR-92) than that of pre-miR-18a, miR-19b and pre-miR-92. A screen of miR-17~92 miRNA expression revealed differing levels of expression among the five different cell lines probed (Fig. S2A and B). In the screen, miR-17~92 expression was highest in HeLa cells, where the relative levels of miRNAs were consistent with the efficiencies in processing observed in the in vitro processing reactions. Relative higher expression of miR-17 and miR-20 is also seen in certain cell lines used in the miRNA expression atlas complied by Landgraf and co-workers29 (Fig. S2C). Mg-dependent folding of miR-17~92. Since all six miRNAs contain the canonical secondary structure features for Drosha

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processing (Fig. S1)18-20 and the hairpins have similar levels of conservation (Fig. S3A),30 we hypothesized that tertiary structure within the miR-17~92 cluster could provide an explanation for the differential processing of the individual miRNAs within this cluster. The miR~17-92 cluster is located within the 3.7 kb intron 3 of the c13orf25 gene.31 To test for structured regions within this RNA, intron 3 was transcribed and folded in the presence of Mg2+, a cation often required for RNA folding.32,33 Digestion of the folded intron with RNase T1 yielded a nuclease resistant product of ~800 nucleotides in length (Fig. 1D). RT-PCR analysis revealed the ~800 nucleotide product to be the miR17~92 cluster, suggesting that the miR~17-92 cluster formed a relatively RNase-resistant structure within the intron. A native gel electrophoretic mobility shift assay (EMSA) 28,34 was used to assess Mg2+ dependent folding of the miR-17~92 cluster under physiologically relevant Mg2+ concentrations.28 The Mg2+ -dependent increase in the mobility of miR-17~92 indicated that the cluster adopts a compact form in the presence of Mg2+ (3–20 mM) (Fig. 1E). Interestingly, in addition to the pre-miRNA hairpins the remainder of RNA sequence of the cluster is highly conserved in vertebrates35 and is predicted to form extensive secondary structure (Figs. S1 and S3), consistent with the idea that this cluster can fold into an evolutionarily conserved structure. Solvent accessibility of individual miRNA hairpins within miR-17~92. We used ribonuclease footprinting36-38 (Fig. 2) to assess the solvent accessibility of the predicted regions of single-stranded RNA7 (Fig. S1) within folded cluster. As expected, cleavages were observed in several of the single-stranded loops and bulges of miRNA hairpins 17, 19a and 20, however, the loops of 18a, 19b and 92 were not cleaved (Fig. 2). These experiments suggest that a large 3' domain of the miR-17~92 cluster containing the miR-19b and miR-92 hairpins is not solvent exposed and is likely protected from enzymatic cleavage through tertiary interactions. Deletion of the 5' region of the cluster exposed the miR-19b and miR-92 hairpins to ribonuclease digestion indicating that the 5' region protects the 3' core domain region within the tertiary structure of the cluster (Fig. 2B and C). This protection is lost upon deleting the miR19b hairpin or the NMSL


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Figure 2. Ribonuclease probing of miR-17~92 structure. (A) Denaturing PAGE analysis of RNAse T1 footprinting reactions of 5' 32P end-labeled miR-17~92. Grey lines indicate positions of size standards generated from an RNase T1 sequencing reaction of 3' 32P end-labeled 3' core domain used to assign approximate positions of RNAse T1 cleavage (black triangles) in the miR17~92 cluster. (B) Denaturing PAGE analysis of RNAse T1 and RNase A footprinting reactions of 3' end-labeled miR-17~92 and 3' 32P end-labeled 3' core domain. The sites of cleavage in the 3' core domain are indicated by white triangles. Cleavage at position 490–497 was assigned using size standards. (C) Predicted secondary structure of miR-17~92 with RNAse T1 cleavages indicated. The position of the mature miRNA sequences are indicated with black lines. The location of the miRNA hairpin stems and loops are indicated schematically beside each of the gel parts. The protected miRNA hairpins are shaded grey. The non-miRNA containing stem-loop (NMSL) within the protected 3' domain is indicated.

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protected from hydroxy radical cleavage37,39 within the wild type cluster, but exposed in the ΔNMSL mutant (Fig. S4). Taken together, the foot printing data suggest that structural elements within the 3' core domain are required to scaffold the folding of the full-length cluster. miR-17~92 adopts a folded, globular structure. In order to directly visualize the folded structure of miR-17~92, we used single particle electron microscopy (EM). Electron micrographs of negatively stained miR-17~92 revealed homogeneous fields of spherical particles ~108 Å in diameter (Fig. 3A). The size of the particle is consistent with the predicted volume for a 260 kDa RNA with a compact globular fold.40 The final 3D reconstruction (Figs. 3B, C, S5 and S6) revealed a globular particle with surface ridges and depressions surrounding a central density. The highly folded structure of the cluster, taken together with the ribonuclease protection data, implies that the core region identified at the 3' end of the pri-miRNA sequence is contained within the central density of the EM reconstruction while the nuclease-accessible miRNA hairpins are likely exposed on the surface. We further examined the importance of the 3' core region to the overall structure of the cluster by analyzing the structure of a 3' truncation mutant by EM (Fig. 3C). This mutant, miR-17~19b, is identical to the full-length cluster but lacks the 3' 67 nucleotides including the miR-92 hairpin. Electron micrographs of negatively stained miR~17-19b revealed that while the mutant particles are globular, they are larger than the wild type cluster particles with a ~137 Å diameter indicating a less compact structure. The 3D reconstruction of miR17~19b illustrated that the mutant does not contain a central density as in the wild type Figure 3. Electron microscopy and single particle reconstruction of miR-17~92 and miR17~19b. RNA (Figs. 3C, S5 and S6). (A) A field of negatively stained miR-17~92 particles from a typical electron micrograph. (B) RepDrosha processing of the core domain. resentative class averages and variance maps for the 3D reconstruction of the full-length miROur Drosha processing and structural data 17~92 cluster. (C) Comparison of the 3D reconstruction of miR-17~92 (blue) with the 3' truncation are consistent with a model in which the mutant miR-17~19b (green). Both reconstructions are contoured to density thresholds that repmiRNA hairpins that are buried within the resent 70% volume recovery for their respective molecular masses (~3σ). This density threshold was chosen to highlight the central density in the structure. Images represent different views of miR-17~92 cluster structure are processed the 3D reconstructions rotated by 90° around the vertical axis as indicated. A 20 Å scale bar and a less efficiently than the miRNA hairpins space filling model of a fourteen base pair dsRNA (PDB ID 1RNA) are included for reference. that are exposed on the surface. Thus, there is an apparent correlation between within the core domain (Fig. S4). Deletion of the NMSL exposed Drosha processing efficiency and surface accessibility of the indiboth miR-19b and miR-92 hairpins to RNase T1 digestion while vidual miRNA-containing hairpins. To test this correlation, we deletion of the miR-19b hairpin exposed the NMSL to diges- assayed the Drosha processing of pre-miR-17 and pre-miR-92 in tion (Fig. S4). In addition, the miR-19b and miR-92 hairpins are the Δ19b cluster where the miR-92 hairpin is solvent accessible


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Figure 4. Structural regulation of miRNA biogenesis. (A) In vitro Drosha processing of miR-17~92 compared to the Δ19b mutant cluster. Northern blotting using probes targeting the mature miRNA sequence detect both the pri- and pre-miRNA. Quantitation of fraction of pre-miRNA generated is plotted versus time to generate kobs values for pre-mR-17 and pre-miR-92 of 0.32 ± 0.08 and 0.079 ± 0.002 respectively. Data for miR-17 and miR-92 generation from wild type miR-17~92 is included for reference. (B) Expression of miRNAs from miR-17~92 and the Δ19b clusters in HEK293T cells. Northern blots using probes targeting the mature miRNA sequence. U6 snRNA is probed as a loading control, a vector expressing human pri-miR-1-1 was used as a transfection control. (C) Expression of the individual miRNAs from the Δ19b cluster is normalized to expression from wild type cluster. miR92a expression shows the highest increase in relative expression. Data is averaged from three independent experiments.

(Figs. 4A and S4). The results demonstrated a dramatic increase in processing efficiency for the miR-92 hairpin rendering it comparable to processing of miR-17 from the mutant and wild type cluster. Taken together with the previous results, these data strongly suggest that tertiary interactions within the intact cluster limit the Drosha accessibility of the miR-19b and miR-92 hairpins. Processing of the miR-17~92 cluster in human cells. To assess the impact of miR-17~92 tertiary structure on processing of the individual miRNAs in living cells, we compared miRNA processing from the full-length cluster, to Δ19b mutant cluster, in HEK293T. The expression of miRNAs 17, 18, 20 and 92 from the mutant cluster, visualized by northern blotting, is increased relative to the wild type cluster. However, the largest increase (1.9-fold) was seen for miR-92 (Fig. 4B and C), suggesting that tertiary structure within the full-length cluster reduces expression of this internalized miRNA relative to the more surfaceexposed miRNAs. It has been reported that Drosha processing is coupled to the subsequent exonucleolytic processing of intronic miRNAs.41,42 Once Drosha removes a miRNA hairpin the remaining primiRNA cleavage product is degraded by the nuclear exosome


and exonuclease Xrn2. Applied to the miR-17~92 cluster, this process could provide a mechanism to generate differential levels of the individual mature miRNAs. In the folded, intact cluster, the outer (non-core) miRNAs are more accessible to Drosha and are therefore more likely to be processed first, followed by degradation of the remaining regions of the cluster. The net result is decreased expression of the 3' core miRNAs (miR-19b and miR-92) relative to the more surface exposed miRNAs (Fig. 5). To test this model, we compared miR-17~92 miRNA expression in cells with or without a shRNA targeting PMscl100, a core protein of the nuclear exosome. Consistent with this model, reduction in PMscl100 protein levels (Fig. S7) increased miR92 expression ~2-fold relative to the non-core domain miRNAs of the cluster (Fig. 5). These data support a model for exosome mediated degradation playing a role in tertiary structure dependent miRNA biogenesis. Structure-dependent miR-92 expression impacts mRNA targeting. Recently, miR-92 has been shown to specifically downregulate integrin α5 (ITGA5) mRNA to suppress angiogenesis.43 We used both end-point (Fig. 6A) and quantitative (Fig. 6B) RT-PCR to compare ITGA5 mRNA levels in HEK293T cells expressing either the intact miR-17~92 cluster or the Δ19b mutant cluster

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Figure 5. Structure mediated differential miRNA biogenesis. (A) Once Drosha processes the surface accessible miRNA hairpins, the nuclear exosome degrades the 3' core Drosha cleavage product, thereby reducing the amount of 3' core miRNAs generated. (B) Comparison of miRNA expression in HEK293T cells transfected with either a miR-17~92 expression vector alone, or co-transfected with a PMscl100 shRNA vector. Northern blotting using probes targeting the mature miR17 and miR92a sequences. U6 snRNA is probed as a loading control, a vector expressing human pri-miR-1-1 was used as a transfection control. (C) miRNA expression with PMscl100 shRNA co-expression is normalized to miR-17~92 expression alone. The PMscl100 knockdown results in a 2.3-fold increase in miR92 expression.

to test the effect of perturbation of the cluster tertiary structure on the expression of this miR-92 target. Expression of the Δ19b mutant cluster resulted in a ~3-fold decrease in ITGA5 mRNA level compared to expression of the full-length miR-17~92 cluster (Fig. 6B). These results indicate that unfolding of the miR-17~92 cluster and the resultant increase in miR-92 processing can result in enhanced repression of a validated target mRNA. Discussion Our work here reveals that tertiary structure within the miR17~92 pri-miRNA cluster, regulates the differential maturation of miRNAs from this cluster, which ultimately impacts the regulation of target mRNAs. A combination of ribonuclease footprinting and EMSA was used to demonstrate that the miR17~92 pri-miRNA folds upon a 3' core domain containing the miR-19b and miR-92 pre-miRNA hairpins and the NMSL. We used single particle electron microscopy (EM) to directly visualize the structures of miR-17~92 and the miR-17~19b truncation mutant. While both RNAs adopt well-defined globular structures, a comparison of the two structures clearly indicates that deletion of the miR-92 hairpin causes an opening of the structure with a striking loss of density in the core of the structure.

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This more open structure induced by mutations in the 3' core domain correlates with an increase in the Drosha processing of the miRNA hairpins that are internalized within the wild type structure. The resulting enhancement of miR-92 levels leads to ~3 fold increased repression of ITGA5, a validated miR-92 target. The effect of differences in miRNA abundances on the scale reported here is illustrated in miR-17~92 mouse models, where miR-17~92 expression in the heterozygous miR-17~92 knock out mouse is ~50% of the wild type results in a significant decrease in embryo weight.3 Interestingly, it has been previously noted that a 3' truncation mutant of miR-17~92 lacking miR-92 induced higher levels of the mature miRNAs than the full-length cluster when expressed in transgenic mice.13 We have shown that disruption of the cluster structure leads to an overall increase in miRNA expression (Fig. 5). Thus, our model provides an explanation for this observation and indicates that structural regulation of miRNA levels within the miR-17~92 cluster likely also functions in the murine system. The differential expression of distinct miRNAs derived from a single cluster has implications for the mechanism of miRNAmediated gene regulation. Myc-activated miR-17~92 expression augments tumor angiogenesis through miR18a and miR19a

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targeting of the anti-angiogenic proteins Tsp1 and CTGF,14 miR17 and miR-20 targeting of the TGFβ tumor suppressor pathway24,44 and miR-17 and miR-20 targeting the E2F transcription factor family.45,46 Myc-activated expression of miR-17~92 leads to a ~5- to 10-fold increase in the 5' miRNAs with only a ~2 fold increase for 3' core miRNA miR92.31 This differential expression is consistent with our findings that tertiary structure within miR17~92 limits the accessibility of miR-92a. Interestingly, miR-92 has an opposite biological function to the 5' miRNAs through a miR-92 mediated repression of pro-angiogenic mRNAs including ITGA5 mRNA.43 Therefore, it is plausible that the suppressed expression of miR-92, through its sequestration within cluster tertiary structure, facilitates the overall pro-angiogenic effect of expression of miR-17~92. Perturbation of miR-17~92 structure by targeting its structural elements could provide new therapeutic strategies for anti-angiogenic therapies.43,47,48 It is plausible that tertiary structure may regulate differential miRNA biogenesis within other miRNA clusters. Like miR17~92, miR-23a~24-2 also expresses miRNAs with opposing biological activities, where miR-27a targets known tumor suppressors,49-51 while miR-24 targets known oncogenes.52,53 Since the miR-23a~24-2 cluster, like miR-17~92, forms a Mg2+ dependent compact fold (unpublished results), it is possible that tertiary structure within the miR-23a~24-2 cluster facilitates the oncogenic activity associated with increased levels of this cluster in certain cancer types.54 Differential expression of miRNAs from a single cluster transcript has also been recently reported for Epstein Barr virus encoded miRNAs (BART miRNAs). It is proposed that tertiary structure within the BART transcripts sequesters certain pre-miRNA hairpins from the Micro-processor resulting in their reduced expression levels.55

Figure 6. (A) RT-PCR analysis of ITGA5 mRNA levels from HEK293T cells expressing the Δ19b mutant compared with miR-17~92 using 5S rRNA as loading control. (B) Comparative quantitative RT-PCR of ITGA5 mRNA from HEK293T cells expressing miR-17~92 or the Δ19b mutant using β-actin mRNA as an internal standard. A comparison of ΔCt from wild type and Δ19b (upper part) gives a ΔΔCt value of 1.48 ± 0.1 which corresponds to a 2.8 fold reduction in ITGA5 mRNA (bottom part). ΔCt values were calculated from six independent measurements.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Materials and Methods

RNA preparation. A T7 RNA polymerase cDNA transcription template encoding c13orf25 intron 3 was generated by PCR (New England Biolabs, Phusion High-Fidelity DNA Polymerase) using human genomic DNA (Roche) as template 5' primer: 5'-GCG CGC TAA TAC GAC TCA CTA TAT GGG AGA GGC CAG CCA TTG GAA GAG CCA CC-3' and 3' primer: 5'-GCG CGC CTC GAG CTG TAA AAT GAA GAT CTC AAA AGT TTT CAG TAA-3'. miR-17~92 was cloned from total HeLa RNA using RT-PCR (forward primer: 5'-GCG CGC GCA GAT CTT AAT GTC AAA GTG CTT ACA GT-3' and reverse primer: 5'-GCG CGC AGG CCT ACC AAA CTC AAC AGG CCG GGA CAA GTG CAA-3') and inserted into a Litmus28i vector to facilitate run-off transcription. Position 518 was chosen as the 5' end of the miR-17~92 3' core domain based on RNase T1 cleavage sites and predicted secondary structure and was cloned into a Litmus28i vector (New England Biolabs) to facilitate run-off transcription. T7 RNA polymerase cDNA transcription templates for the mutants ΔNMSL, Δ19b and miR-17~19b were prepared by PCR (Invitrogen® Platinum Pfx DNA Polymerase). In vitro drosha miRNA processing. RNAs were annealed using the following protocol: 90° C 30 sec, 70° C 1 min, then slow cooled to 25° C over 20 min in 10 mM sodium cacodylate pH 6.8,


10 mM NaCl, 10 mM MgCl2 and 0.1 mM EDTA. Drosha and DGCR8 were overexpressed in HEK293T cells and immunoprecipitated.25 Processing reactions (108 μL) were performed on annealed RNA (185 nM final) in a 50% Drosha bead slurry with 6.4 mM MgCl2 and 1.33 U/μL Superase Inhibitor (Ambion). Reactions were quenched by phenol/chloroform extraction followed by ethanol precipitation, resuspended in 8 M urea and denatured at 95° C for 2 min and then placed on ice (to ensure complete denaturation of the RNA), resolved by denaturing (8 M urea) 15% (19:1) PAGE. Cleavage products were resolved by 15% denaturing PAGE and electrotransferred to GeneScreen Plus (ABI) and hybridized with UltraHyb Oligo buffer (Ambion). Blots were probed with 32P 5' end-labeled DNA oligonucleotides (42°C) complementary to the mature miRNA sequences. kobs values were obtained from plots of fraction of mature pre-miRNA versus time fit to a first order exponential (GrapghPad 5.0). RNA folding. RNAs were annealed using the following protocol: 90° C 30 sec, 70°C 1 min, then slow cooled to 25° C over 20 min in 10 mM sodium cacodylate pH 6.8, 10 mM NaCl, 10 mM MgCl2 and 0.1 mM EDTA. RNAs annealed without Mg2+ were treated similarly, except 1 mM EDTA was used in place of 10 mM MgCl2. RNA folding was assayed using agarose gel electrophoresis (2.5% agarose, 2.5 mM MgCl 2, 100 mM tris-glycine pH 8.0 for Mg2+ native gels and 1% agarose, 2 mM EDTA, 100 mM tris-borate pH 8.0 for non-magnesium gels) and RNAs were visualized by ethidium bromide staining. RNase T1 digestion. Intron 3 RNA (35 μg) was renatured and digested with 67 U RNase T1 (Ambion) for 1 min at room

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temperature. The reaction was quenched by phenol/chloroform extraction and ethanol precipitation and cleavage products resolved by 6% (29:1) 8 M urea denaturing PAGE. 5' end-labeling was performed with γ 32P ATP (Perkin Elmer, 6,000 Ci/mmol) and T4 polynucleotide kinase (New England Biolabs). 3' end labeling was performed with 5' 32P pCp (Perkin Elmer, 3,000 Ci/mmol) and RNA ligase1 (New England Biolabs). RNase T1 protection (footprinting) reactions (100 μL, 0.05 U RNAse T1 (Ambion)) were performed on 5' or 3' 32P end-labeled magnesium annealed miR-17~92, 3' 32P end-labeled magnesium annealed miR-17~92 3' core domain, 3' 32P end-labeled magnesium annealed ΔNMSL and Δ19b mutants. After the indicated times the reactions were quenched by phenol/chloroform extraction and ethanol precipitation. Cleavage products were resolved by 6% (29:1) 8 M urea denaturing PAGE. Sites of cleavage were assigned by comparison to RNase T1 sequencing reactions and RNA size standards. An RNase T1 cleavage site was defined as a band or region having a greater than two-fold increase in intensity over the same band or region in the no RNase T1 treatment lane. Electron microscopy. Annealed RNAs (0.2 mg mL -1) were applied to carbon-coated and glow-discharged 400 mesh Cu/ Rh Maxtaform grids (Ted Pella, Inc.). The grids were subsequently washed with 16% ammonium molybdate and 2% uranyl acetate. Grid preparation using only ammonium molybdate or uranyl acetate revealed no significant differences in the appearance of the RNA particles. The grids were imaged under low electron dose conditions on a Tecnai F20 electron microscope set to 200 keV (FEI, situated in the Microscopy and Imaging Facility, University of Calgary, Alberta, Canada). The images were recorded at 50,000x magnification on Kodak SO-163 film over a defocus range from -1 μm to -2.5 μm. The micrographs were digitized (Nikon Super Coolscan 9000) and pixel averaging was applied to reach a resolution of 5.08 Å/pixel. Image processing and single particle reconstruction made use of both EMAN56 and SPIDER57 software packages for parallel processing of the data. miR-17~92 particles (17,120 in total), and miR-17~19b particles (6,574 in total) were selected from the micrographs using semi-automatic boxing methods (boxer, EMAN). The 3D reconstruction of the miR-17~92 cluster will be described, though a similar procedure was used for both RNAs. The particles were centered and masked. The images were then aligned and subjected to reference-free classification and averaging. For miR-17~92, 175 class averages were generated each from EMAN and SPIDER (350 independent class averages). The two sets of class averages were combined and reduced into a common set of 45 class averages generated from a subset of the miR-17~92 particles (12,880 particles selected based on self-clustering during the classification process). Euler angles were assigned to the class averages in EMAN and SPIDER and initial 3D models were constructed using common lines in Fourier space. The initial models were refined through 5 iterations, and both reconstructions were then low pass filtered to 20 Å resolution, aligned and averaged together. Projections of this averaged model were made to initiate a round of reference-based classification, alignment, initial reconstruction, refinement and averaging. This last

step was repeated to generate the final model, and an identical procedure was used for reconstruction of the truncation mutant, miR-17~19b. The distribution of class averages that contributed to the final structures was found to be uniform around the Euler sphere. The robustness of this 3D reconstruction procedure was tested by using incorrect starting models for reference-based reconstruction of both miR-17~92 and miR-17~19b (Fig. S6). The handedness of the 3D reconstructions was arbitrarily assigned and does not affect the conclusions drawn from the structures. The resolution of the final reconstructions was determined to be 21 Å for miR-17~92 and 27 Å for miR-17~19b, by calculating the Fourier shell correlation (FSC) 58 between two independent half data sets using the 0.5 FSC criterion. Particle sizes were determined by iterative centering and aligning the entire particle sets (EMAN, cenalignint). Finally, the reconstructions were contoured to their correct molecular volumes for viewing in Chimera.59 In vivo drosha miRNA processing. hsa-miR-1-1, miR-17~92 and Δ19b constructs were inserted (KpnI and XhoI sites) into pcDNA 3.1 (+). HEK293T cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS). Cells were transfected with 30 μg of total DNA (5 μg miR-1-1 vector plus 25 μg cluster vector; or 5 μg miR-1-1 vector plus 12.5 μg cluster vector plus 12.5 μg PMscl100 shRNA vector) using the calcium phosphate method.60 Total RNA was prepared with 1 mL of Trizol (Invitrogen). 20 μg of total RNA was resolved by 15% denaturing PAGE and electrotransferred to GeneScreen Plus (ABI) and hybridized with UltraHyb Oligo buffer (Ambion). Blots were probed at 42°C (37°C for cell line screen Fig. S2) with 32P 5' end-labeled DNA oligonucleotides complementary to the mature miRNA sequences (except for miR-19a and miR19b where the hairpin terminal loops were targeted). The human miR-1-1 pri-miRNA insert was generated by PCR: forward 5'-ATA CCG CTC GAG CTT CTG CCT TTC TGG ATC GTG T-3'; reverse 5'-ATA CCG CTC GAG CTG CTG ACA CAG GCA AAG TGA C-3' and was cloned into the XhoI site of pcDNA 3.1 (+). The PMscl100 shRNA61,62 insert was generated using overlapping PCR primers: forward 5'-GCG CGC AAG CTT TGC TGT TGA CAG TGA GCG ACA AGA GTA CAG ATG ATC ATG TTG TGA AGC CAC AGA TGT A-3'; reverse 5'-GCG CGC CTC GAG TCC GAG GCA GTA GGC AAA GAG TAC AGA TGA TCA TGT TTA CAT CTG TGG CTT CAC AAC ATG ATC ATC-3' and was cloned into the HindIII and XhoI sites of pcDNA 3.1 (+). RT-PCR. End point semi-quantitative RT-PCR (Invitrogen Superscript One-Step RT-PCR with Platinum® Taq) was performed using 10 ng of total RNA as template. qRT-PCR was done in two steps. cDNA was synthesized (Invitrogen SuperScriptTM III Reverse Transcriptase, 30 min at 50°), using 5 μg of total RNA template; cDNA was then quantified by qPCR (Invitrogen SYBR® GreenERTM qPCR SuperMix Universal) using a RotorGene RG-3000 (Corbett Research). qRT-PCR primers: ITGA5 forward, 5'-CGT GTG GTT TTA GGT GGA-3'; ITGA5 reverse, 5'-TCC CCT GAG AAG TTG TAG AG-3'; β-actin forward, 5'-AGG CAC CAG GGC GTG AT-3'; β-actin reverse, 5'-GCC CAC ATA GGA ATC CTT CTG AC-3'.


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Acknowledgments

We thank D.C. Arthur and R.A. Edwards for helpful discussions on experimental methods and manuscript preparation; C. Leung for assistance with figures. This research was supported by grants from the Canadian Institutes of Health Research (CIHR), Canadian Cancer Society and the Howard Hughes International Scholar program to J.N.M. Glover; a grant References 1.

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Supplemental material can be found at: www.landesbioscience.com/journals/rnabiology/article/17410

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