Functional validation of microRNA-target RNA interactions

Methods 58 (2012) 126–134

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Methods journal homepage: www.elsevier.com/locate/ymeth

Review Article

Functional validation of microRNA-target RNA interactions S. Vasudevan Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, United States

a r t i c l e

i n f o

Article history: Available online 14 August 2012 Communicated by Gary Brewer Keywords: MicroRNA Target Base-pairing

a b s t r a c t MicroRNAs are small, non-coding RNA regulators of gene expression with important outcomes in cell state, proliferation, metabolism, immunity and development; their deregulation leads to significant clinical consequences. MicroRNAs and their associated target RNAs can be identified by genetic, bioinformatic and biochemical methods. MicroRNAs can recognize target mRNAs via direct base-pairing and recruit effector complexes to modulate their gene expression in a sequence-specific manner. MicroRNA interactions with target RNAs produce their roles in gene expression. The following are some of the validation methods employed to confirm functionally relevant microRNA interactions with their target mRNAs. Each method involves interference with the microRNA or the target mRNA to disable their interaction, which should lead to loss of microRNA-mediated gene expression if the interaction is functionally consequential. Subsequent alleviation of the interference and restoration of productive base-pairing interactions between the microRNA and target should rescue microRNA-mediated gene expression and confirm the functional requirement for direct microRNA-target mRNA interaction. Characterization of functional microRNA interactions with their target mRNAs will provide significant insights into their gene expression regulatory mechanism and lead to the development of potential therapeutic approaches to manipulate these interactions and their consequent gene expression outcomes. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction MicroRNAs are small 19–24 nt regulatory non-coding RNAs that usually recognize their target sequences via base-pairing and direct their associated effector complex to the mRNA to mediate downregulation and upregulated expression in specific conditions. The relative levels of microRNA compared to that of mRNA influences the magnitude of gene expression regulation; from small adjustments to substantial changes in gene expression. The expression and levels of different microRNA families may be regulated and produce a combinatorial modulatory effect on target mRNAs that bear multiple microRNA sites, thus adding greater adaptability to gene expression control [1–3]. The Argonaute (AGO) or eukaryotic initiation factor 2C (eIF2C) family of proteins bind microRNAs to form the microRNP (microRNA-associated protein complex), which is directed to target RNAs in a sequence specific manner and regulates mRNA expression. The nature of the sequencespecific interactions of microRNAs with other RNAs as targets (usually the untranslated regions (UTRs) of mRNAs but also coding regions and non-coding RNAs) determines gene expression outcomes. Complete base-pairing of the microRNA to its target leads to mRNA cleavage, degradation and repression, determined by the functional characteristics of the AGO family member present [1,2,4,5]. Depending on the microRNP-associated effector proteins E-mail address: [email protected] 1046-2023/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2012.08.002

such as GW182 or FXR1-iso-a [6,7], partial base-pairing to target sites by the microRNP leads to the predominantly observed gene expression downregulation by mRNA deadenylation [8–10] and translational repression [6] (GW182-bearing microRNPs), or to upregulated gene expression, by translation activation (FXR1-isoa-associated microRNPs in specific conditions with distinct mRNAs) or abrogated microRNA-mediated repression (relief of repression) [6,11,12]. Candidate microRNAs are identified by genetic (such as screens with depletions and overexpression), bioinformatic (such as database, seed sequence/UTR analyses) or biochemical analyses (copurifications with mRNAs or microRNP/AGO complexes) [13–19]. The physical interactions of microRNAs with target RNAs or RNP complexes can be demonstrated by biochemical purification with in vivo crosslinking coupled microRNP, aptamer-tagged mRNA or biotin-tagged microRNA affinity purification as described previously [17–22]. Validation of functionally productive microRNAtarget RNA interactions is required to demonstrate that the association of microRNAs with their specific targets leads to the regulatory outcomes on gene expression and whether the regulation of gene expression is direct via sequence-specific, base-pairing recognition of the target or indirect via the effects of other directly targeted genes. The methods used to confirm the regulatory significance of specific microRNA-target associations involve interference with microRNA-target interaction, which should result in loss of microRNA-mediated expression. Subsequent abrogation of

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the interference and recovery of base-pairing between the microRNA and target should restore microRNA-mediated expression and confirm the functional outcome of direct microRNA-target mRNA interactions on gene expression. Three approaches to validate microRNA-target mRNA interactions are:  Interference with microRNA levels by depletion of pri-/premicroRNAs followed by complementation analysis of loss of function with mature microRNAs (Protocol A)  Blockage of the microRNA target site on the mRNA with antisense (LNA or 20 -O-methyl oligonucleotide) protectors (Protocol B)  Disruption of microRNA-target mRNA base-pairing via mutational alterations of the microRNA target site on the mRNA, followed by complementation analysis of loss of function with compensatorily mutated microRNAs (Protocol C) 2. Interference with microRNA levels by depletion of pri-/premicroRNAs followed by complementation analysis of loss of function with mature microRNAs (Protocal A)

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oligonucleotides (oligos) that are subsequently annealed as described below [38,39]. ShRNAs instead of siRNAs and microRNAs can be expressed from transfected vectors that are commercially available (pSM2 for constitutive expression; pTMP or pTRIPZ for tetracycline inducible expression, Open Biosystems). The shRNA or microRNA is processed from these transfected vectors in vivo, expressed by Polymerase II as an abundant pri-microRNA using the basic primiR30 structure and sequence with replacement of the microRNA and microRNA⁄ sequences with the sequences of the siRNA (or rescue microRNA or control versions) and the complementary sequence, respectively (as described in [40,41]). Careful titrations are required to ensure that the synthetic microRNAs are not overor under-expressed, which could lead to additional spurious effects or lack of rescue, respectively. The siRNA and rescue microRNA must be titrated carefully at the lowest levels (between 50 and 200 nM, the ideal concentration in our experiments is 100 nM) [38] as excessive levels of small RNAs may lead to interference with endogenous small RNA functions since free unbound AGO is limiting in the cell [42]. 2.3. Annealing oligonucleotides to form duplexes

2.1. Approach MicroRNAs can be depleted to cause loss of function by knockdown of the pri- or pre-cursor forms (pri- or pre-microRNA) with siRNAs/shRNAs followed by rescue of any loss of function with addback of synthetic mature microRNA [11,23] (Protocol A, Fig. 1A and B). The siRNA/shRNA is designed to target regions of the pri or pre-microRNA that do not overlap with the mature microRNA sequence. This method confirms requirement of microRNA function for regulated target mRNA expression but does not validate direct microRNA-target RNA interactions. The advantage of this method is that any loss of function due to interference with microRNA levels can be further affirmed by complementation analysis with addback of the mature microRNA. Recovery of microRNA function confirms the requirement (direct or indirect) for the specific microRNA (Fig. 1B). 2.2. Design The pre-microRNA loop regions, which do not overlap with the microRNA sequence, is targeted with siRNAs or shRNAs (Fig. 1B2 and 3). If the loop region is AU-rich or overlaps with part of the microRNA, the pri-microRNA can be targeted instead at suitable regions that do not overlap with the microRNA. Blocking the loop region can lead to loss of the mature microRNA, which may be a result of siRNA-mediated decay of the pri-/pre-microRNA and/or blocked processing since the loop region plays important regulatory roles in microRNA processing in several cases [24–33]. The consequent loss of function can be complemented by titrating back synthetic candidate or control mature microRNAs. The rescue microRNAs are synthesized as a duplex siRNA or mature microRNA mimic duplex. A 50 -phosphate is necessary for the microRNA/siRNA strand to be recruited into RISC from a duplex. Therefore, the microRNAs and siRNAs synthesized are phosphorylated at the 50 end on the target-complementary strand, which reduces selection of the other non-phosphorylated strand and thereby, off-target effects [34–37]. Since the siRNA/shRNA targeting the pri- or premicroRNA does not overlap with the mature microRNA sequence, the added back compensatory, mature microRNAs are resistant to the siRNA/shRNA and can functionally replace the depleted microRNA in a dose dependent manner. SiRNAs or microRNAs, phosphorylated at the 50 -end of the target-complementary strand of the siRNA or the microRNA strand of the added back microRNA duplex, can be ordered as duplexes or as individual RNA

The RNA oligos are either synthesized as individual sense and antisense, 50 phosphorylated (on the microRNA or target-complementary siRNA strand) RNA oligos (IDT biotechnologies, IA) or as similarly phosphorylated duplexes [34–37]. The individually synthesized RNA oligos are then resuspended at 200 pmol/ll in annealing buffer (10 mM Potassium Acetate (KOAc) and 1 mM EDTA) and measured. The RNA oligos are then mixed in equimolar proportions, heated to 95 °C for 5 min and allowed to slow cool to room temperature to anneal the two strands. If a duplex is commercially synthesized, it is resuspended in RNase-free water or the annealing buffer and measured. The duplexes are then transfected as described. 2.4. MicroRNA-mediated regulation Repression/downregulation can be observed in asynchronously growing cells. We find that repression with our reporters is enhanced when the cells are at least partially synchronized and replated/detached with trypsin and restarted in the cell cycle in fresh growth factor (10% serum, L-glutamine and DMEM) media and grown to a common phase, in particular, the S/G2 phases (from quiescent confluent or serum-starved quiescent cells to a normal growing 50% confluency culture at 5–10  105 cells/ml in a 10 cm plate in fresh growth factor media with the cells contacting each other but with sufficient space to stimulate rather than inhibit growth) [43]. Alternatively, growing cells can be partially synchronized (without arresting them) when they are replated/detached with trypsin and restarted in the cell cycle at high densities in fresh growth serum media (from 5–10  105 cells/ml in a 10 cm plate to at least 1  106 cells/well of 24 well plates) to stimulate proliferation (induced by the cell to cell contact of growing cells in fresh growth factor containing media at high densities) [44]. It has been demonstrated that GW182, the key repressor of the microRNA pathway [6], is responsive to the cell cycle, where the GW bodies (a consequence of repression [45]) alter with the cell cycle. The bodies are least observed or intense in G0 and also in G1 and maximum at S/G2 phases of synchronous growth [46]. Synchronizing cells and restarting the cell cycle to grow to the S/G2 phase may therefore, enable greater repression in some cases. 2/ 3 of the cells of an asynchronous population are in G1 [47,48], suggesting that repression may be reduced here for some cases, consistent with the decline of GW bodies and alteration of GW182

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A

B

Fig. 1. Protocol A. Interference with microRNA levels by depletion of pri-/pre-microRNAs followed by complementation analysis of loss of function with mature microRNAs MicroRNAs can be depleted to cause loss of function by knockdown of the pri- or pre-microRNA with siRNAs/shRNAs followed by rescue of any loss of function with addback of synthetic microRNA [11,23]. (A) Protocol timeline. (B) (1) The pre-cursor, pre-mir369 encodes two microRNAs, miR369-5p shown in orange and miR369-3p shown in blue. 50 P = 50 phosphate. The mature miR369-3p is synthesized and base-pairs with and alters the expression of a TNFa ARE reporter. (2) The pre-miR369 can be targeted with an siRNA (shown as a black curved line), which base-pairs with the loop region of the pre-microRNA but does not overlap with the mature miR369-3p sequence on the premicroRNA. This leads to loss of processing and/or degradation of the pre-miR369, leading to depleted mature miR369-3p levels and thereby, loss of gene regulation of the TNFa ARE reporter (depicted by a dotted arrow). (3) The loss of mature miR369-3p levels and function due to interference with its pre-microRNA as described in (2) can be complemented by adding back in vitro synthesized, miR369-3p as a duplex (black lines), which is 50 phosphorylated (50 P) on the miR369-3p strand to restore the depleted microRNA levels (shown in black). Restoration of the mature microRNA leads to rescue of microRNA-mediated gene regulation of the TNFa ARE reporter, confirming that the tested microRNA is required (directly or indirectly) for regulation of gene expression of the target, TNFa ARE.

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[46]. Therefore, repression can potentially be enhanced by a combination of synchronization and then release into active proliferation by replating cells to restart the cell cycle (Fig. 1A) [43] or by stimulation of proliferation by plating growing cells in fresh growth media at high densities [44]. Upregulation was observed with selected reporters in the quiescent or G0 state [7,11,20,43,49–54]. Quiescence is an assortment of states and difficult to distinguish easily from the alternative options of G1 arrest or senescence [55–57]. Specific mRNAs expressed or mobilized in quiescence are upregulated [7,49–54]. Activation is observed with mRNAs that are usually mobilized in quiescence, such as TNFa and KLF4 mRNAs, associated with some select microRNAs increased in G0, likely because of their specific recruitment into an alternative microRNP (with altered interactions with the repressive GW182 cofactor) [46,49,58–61], while pre-existing mRNAs associated with the proliferation state, such as Cyclin E and DEK, are downregulated [7,50,62,63]. Upregulation of specific mRNAs and downregulation of proliferation-associated transcripts lead to selective gene expression that contributes to maintaining the quiescent state [7,46,49,50,58–63]. 2.5. Protocol A The protocol described (Fig. 1A) is performed using HEK293 cells and a previously constructed (Firefly Luciferase bearing the TNFa AU-rich element (ARE) driven by the CMV promoter) ARE reporter. Renilla Luciferase construct (CMV or b-actin promoter) is co-transfected as a normalization control [11,20]. The Firefly test reporter was also cloned under a tetracycline inducible CMV promoter for inducible, regulated expression. The ARE reporter along with the Renilla vector can be transfected along with pLVX-teton advanced (Clontech) which constitutively expresses tetracycline Activator (rTA). Firefly expression can be induced with the addition of 50–100 ng/ml of doxycycline; the doxycycline concentration and duration can be manipulated to ensure that the reporter to microRNA ratio is at equimolar or higher levels of the microRNAs. The microRNA, miR369-3p that can target this reporter is detected in HEK293 cells in confluent G0 conditions and in the S/ G2 phases of the cell cycle. The pre-microRNA loop region is targeted by an siRNA, which leads to a loss of microRNA levels and function. Addition of synthetic microRNA duplex comprising annealed miR369-3p and its antisense rescues the loss of function [11]. 1. Grow fresh HEK293 cells at passage 3 of freshly thawed cells in DMEM with 10% FBS, L-glutamine and Penicillin/Streptomycin antibiotics. Pass the cells to maintain logarithmically growing cells, 24 h prior to transfection and cell cycle manipulations. 2. Synchronized cycling and quiescent cells: While repression can be observed in asynchronous growing cells, we find that repression with our reporters is enhanced when the cells are synchronized, detached with trypsin/replated to restart the cell cycle in fresh growth factor media from (a) confluent arrested cells [43] or (b) serum-starved quiescent arrested cells [43] or (c) from growing cells that are stimulated to proliferate by detaching and replating them at high densities in fresh growth factor media [44]. (a) The cells are taken from confluent quiescent plates (grown to confluency and further grown till growth factors are depleted in the media and the cells are quiescent, Fig. 1A), trypsinized and replated in fresh media at less confluent densities (5–10  105 cells/ml per 10 cm plate where they are contacting each other but do not inhibit growth due to plenty of space and growth factors in new media, which stimulate their growth) to restart the cell cycle [43].

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(b) If serum-starvation is used to arrest and synchronize cells, the cells are taken from the sparsely populated, serumstarved quiescent plates (less than or equal to 1  105 cells/ml per 10 cm plate), trypsinized and replated in fresh media at higher densities (5–10  105 cells/ml per 10 cm plate where they are contacting each other but do not inhibit growth due to plenty of space and growth factors in new media, which stimulate their growth) to restart the cell cycle [43]. (c) To obtain enhanced repression with growing cultures without growing them to confluency or serum-starvation, actively growing cells (that were not confluent) can be replated in fresh growth factor containing media at high densities (at least 1  106 cells/well of 24 well plates). These cells actively proliferate and are not in the quiescent state, which is induced after persistence at high confluency upon growth factor depletion and contact inhibition [44,47,48,55,57]. Depending on the cell line, cells can be synchronized by double thymidine block (HeLa cells) or by serum-starvation (HeLa and some HEK 293 cells at low densities of less than or equal to 1  105 cells/ml per 10 cm plate) or growth to confluent quiescence (effective procedure for HEK293 cells described below) [43]. For growth to confluency to synchronize cells (for HEK293 cells), the cells can be analyzed as quiescent cells for upregulation of certain targets. Alternatively, after quiescence-induced synchronization, the synchronized cells can be restarted into the cell cycle and grown to the S/G2 phases to observe repression as described below (Fig. 1A). The cell cycle phase was monitored by harvesting a duplicate sample every 6hrs by propidium iodide staining and fluorescence activated cell sorting (FACS) analysis [47,48,55,57]. 3. Plate growing cells at 1  106 cells/well of 24 well plates. Allow cells to grow to confluency, until the media turns yellow (about 72 h, depending on initial densities and growth rate) (Fig. 1A). 4. The confluent cells were transfected with the siRNA duplex or siRNA/shRNA vector for at least 30–36 h before the end of the 72 h period. The siRNA is usually transfected as double stranded RNA at 50–100 nM. If an shRNA vector (pSM2 for constitutive expression; pTMP or pTRIPZ for tetracycline inducible expression; Open Biosystems [40,41]) is used to express the same siRNA sequence of the duplex, we use 250–500 ng per transfection. If the inducible vector is used then 1 lg of pLVX tet-on advanced plasmid (Clontech) to express the tetracycline activator (rTA) constitutively, is also transfected. The transfection is carried out using nucleofection or trans-it-293 transfection reagent (MIRUSBIO), following the manufacturer’s instructions. Eighty to eighty-five percentage transfection efficiencies can be achieved with these transfection methods and HEK293 cells. The added back rescue and control microRNAs can be transfected at the same time as the reporters at 50–100 nM in a similar manner or subsequently, 30 h prior to harvest as described below (Fig. 1A). The reporters (50 ng of Firefly-test UTR/ARE reporter and 20 ng of Renilla co-transfection control) are transfected using nucleofection or trans-it-293 transfection reagent (MIRUSBIO) at the end of the 72 h period or transfected simultaneously with the siRNA/shRNA. A control Firefly-control UTR/mutant ARE reporter was transfected in parallel, control experiments (Fig. 1A). If tetracycline induction of the reporter was used, then 50 ng of the tetracycline inducible Firefly-test reporter plasmid, 20 ng of Renilla co-transfection control plasmid as well as the tetracycline Activator (rTA, 1 lg of pLVX-teton advanced (Clontech), which constitutively expresses rTA) were

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transfected along with the siRNA/shRNA or at the end of the 72 h period. The test Firefly reporter was induced with 50–100 ng/ml of doxycycline (Sigma), at the end of the 72 h period. 5. After the 72 h growth to confluency, the cells were further allowed to grow and metabolize the remaining nutrients and growth factors in the same media for another 36–48 h (total = 72 h + 36 or 48 h, depending on initial densities and growth rate) for further depletion of growth factors in the media and confluency leading to quiescent cells [11,43,50,53,54]. This step can be reduced to 24–36 h for cells that are eventually replated for 12 h in fresh media for synchronized, restarted cycling S/G2 cells (total = 72 h + additional 36 or 48 h, inclusive of 12 h after replating in fresh growth factor containing media) but is required to be as extended as much as possible to obtain quiescent cells. Both the quiescent as well as the replated cells are harvested at the same time (Fig. 1A). During this period, the added back rescue microRNA or a control microRNA, synthesized in vitro or as an shRNA vector, is transfected (and induced if under doxycycline control) for at least 30 h before the end of the 36–48 h period to restore microRNA levels and complement the loss of microRNA function that were depleted by the siRNA (Fig. 1A and B1–3). A control microRNA is transfected instead, in parallel cells, where the microRNA levels remain depleted and the loss of microRNA function is not functionally complemented as a control. 6. At the completion of this additional period, when the cells are fully confluent and the media depleted of growth factors, the cells are harvested as confluent quiescent cells to observe upregulation of specific targets (Fig. 1A, G0 cells) [11,43,50, 53,54]. Alternatively, to observe repression with synchronized restarted cycling S/G2 cells, the confluent cells were detached with trypsin and replated with fresh media (at non-confluent but not sparsely spread densities, 5–10  105 cells/ml per 10 cm plate where they are contacting each other but do not inhibit growth due to plenty of space and growth factors in new media, which stimulates their growth) to restart the cell cycle [11,43,50,53,54]. At about 12 h after replating with fresh media, the cells can be observed to be more in the S/G2 phases of the cell cycle by FACS, at which point the cells are harvested (Fig. 1A, S/G2 phase cells). 7. The harvested samples are analyzed for microRNA-mediated expression by luciferase assays (following the manufacturer’s instructions, Promega). The samples are also analyzed for RNA levels, examining the levels of the reporters to check for microRNA-mediated alterations of target RNA levels as well as the levels of the targeted microRNA by RNase Protection Analysis (RPA) or qRT-PCR [11,43,50,53,54]. RNA samples are extracted in lysis buffer (Hepes pH 7.0, 100 mM NaCl, 6 mM MgCl2, 0.025% NP40 and RNase inhibitor), followed by proteinase K digestion in proteinase K digestion buffer (300 mM NaCl, 1.5% SDS, Tris pH 7.0), Trizol (Invitrogen) extracted and precipitated with NaOAc and 30 lg of glycogen to facilitate RNA extraction for RT-PCR analysis. 2.6. Controls (1) Depletion of the endogenous microRNA and complementation with re-expression of the microRNA will also affect expression of the endogenous target mRNA, which can be analyzed by Western blotting in parallel with the reporter assay and should show a similar response. (2) AGO2/microRNP purification using AGO2 antibody (cat#07-590, Millipore or anti-AGO2 clone 4G8 from Wako) to immunoprecipitate AGO2-associated targets followed by RT-PCR analyses can be performed to ensure decreased target mRNA association with AGO2 upon depletion of the microRNA

and subsequent restoration of the target mRNA association with AGO2 upon re-expression of the microRNA but not when the control microRNA is expressed instead [11]. 2.7. Potential issues (1) The ratio between the microRNA and reporter is critical and must be titrated carefully [3]. The reporter RNA levels can be kept limited or induced briefly under the tetracycline inducible promoter by manipulating doxycycline concentrations and durations to ensure that the ratio of microRNA to reporter mRNA is higher. In the oocyte system, we calculated a 4:1 ratio for a reporter with four target sites [49]. (2) Additionally, increasing siRNA and microRNA levels excessively (higher than 200 nM, the ideal concentration in our experiments is 100 nM) is refractory [38] because of the limiting unbound AGO available in the cell to form functional microRNPs with the additional exogenous siRNA and microRNA [42]. (3) siRNA and microRNA stability may be low. The small RNA sequence can be altered with modified nucleotides (such as 20 -fluoro modified nucleotides) to impart more stability [37,64,65], used at higher levels or expressed from inducible vectors. (4) Effects due to overexpression can be eliminated by titrating the microRNA, comparing levels of the exogenously expressed microRNA to that of the endogenous. (5) Any off-target effects of the siRNA/microRNA should be eliminated by titrating to lower concentrations at which a scrambled control does not cause effects. (6) Quiescence [11,20,43,49] is transient and less frequently observed and it is also influenced by cues such as cell to cell contact [44,47,55,66]. Inducing quiescence may not be possible in all cell lines. A block to cell division by confluency or serum-starvation and other methods in various cell lines leads to cell death, G1 arrest or senescence rather than quiescence in cells lacking the ability to enter quiescence, which, in the absence of specific tests and markers to distinguish G0 from G1 arrest and senescence, can misleadingly suggest G0. Additionally, these procedures lead to extensive cell death, which can affect readings of microRNA-mediated gene expression [11,20,43,47,49,55,57,66]. 3. Blockage of the microRNA target site on the mRNA with antisense (LNA or 20 -O-methyl oligonucleotide) protectors (Protocol B) 3.1. Approach The candidate microRNA functions can be blocked away from its target by the use of modified oligonucleotides that basepair and sequester the microRNA, called antagomirs [51,67], or with RNA sponges [68] that mimic the target site and are overexpressed to sequester the microRNA away from the target. Alternatively, as discussed below, target protectors, developed first by Giraldez, Schier et al. [69] to block target sites rather than the microRNA and observe loss of microRNA function on a specific target can be used (Fig. 2). Specificity is imparted by the protector complementary not only to the target site but also to unique sequences adjacent to the target site. By targeting the specific mRNA target site (Fig. 2a and b) and not the microRNA (Fig. 2a and c), only the tested target mRNA expression will be affected and analyzed. Other genes and pathways controlled by the microRNA, which might cause indirect gene expression readouts, will not be affected by the protector. Because the protector can be designed specifically against each of several target sites via their unique adjacent sequences within the same mRNA UTR [49,69], this method allows functional determination of the critical target site for a given mRNA with multiple sites. Mutated versions of the protector can be used as controls that would fail to block target-microRNA base-pairing.

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Fig. 2. Protocol B. Blockage of the microRNA target site on the mRNA with antisense (LNA or 20 -O-methyl oligonucleotide) protectors Target protectors can be used [69] to block target sites rather than the microRNA and observe loss of microRNA function on a specific target. Specificity is imparted by the protector complementary not only to the target site but also to unique sequences adjacent to the target site. Only the tested target mRNA expression will be affected and analyzed while multiple genes and pathways controlled by the microRNA will not be affected. (a) Base-pairing of the target sites on the TNFa ARE reporter with miR369-3p microRNA leads to regulated gene expression of the reporter at the two specific target sites shown. (b) The selected target sites and the unique sequences adjacent to these specific target sites (27 nts or more, not shown to scale) can be blocked [49,69] with modified (N = Locked nucleic acid, LNA or 20 -O-methyl) [67,72,73] antisense to prevent microRNA, miR369-3p base-pairing. This should abrogate regulated expression of exclusively the TNFa ARE reporter, only if the selected target sites to be blocked are the critical regulatory target sites on this mRNA. (c) Alternatively, the microRNA, miR369-3p, can be directly blocked with antisense antagomirs [67](N) that are complementary to the microRNA (partially or completely to the full-length microRNA) to prevent regulated expression of all its direct targets including, but not restricted to, the TNFa ARE reporter.

Protectors can also alter the RNA/RNP conformation as well as prevent RNA binding protein interactions with the target [70,71], in addition to blocking microRNA target sites that are functionally critical. Therefore, additional tests are required to distinguish between loss of mRNA/mRNP conformation or RNA binding protein-target interactions and true loss of direct microRNA-target base-pairing interactions at the defined site. 3.2. Design The target protector is designed as an antisense that can basepair with the 7–8 nucleotide target site complementary to the microRNA seed sequence and about 10–20 nts on either side of this sequence (or about 20 nts on one side alone), specifically at the site chosen to be targeted against by the protector [49,69] (Fig. 2b, 27 nts or more, not shown to scale). Locked nucleic acids (LNAs) that impart specificity and enhance affinity for complementary sequences, are added at the 4th or 5th nt and then every 3rd nt position on the antisense target protector [49] or at least three positions on each end of the entire protector [67,69]. The rest of the antisense is modified with 20 -O-methyl nucleotides to render the oligonucleotide stable [49,67,69,72,73]. Alternatively, the entire oligonucleotide is only 20 -O-methylated without LNAs. 20 -Omethylated oligos can function efficiently in the nucleus as well as in the cytoplasm as suggested by antisense techniques [72,73]. A control LNA oligo is also derived by scrambling the sequence. LNA is a nucleic acid analog that contains a 20 -O-40 -C methylene bridge, which restricts the flexibility of the ribofuranose ring and locks the structure into a rigid C3-endo conformation, greatly enhancing hybridization and stability [64,65,74,75]. The nucleotides that are not LNA modified are 20 -O-methylated RNA, which enhance stability and hybridization and prevent RNase H activation. Any mRNA degradation due to base-pairing with the protector would be due to RNase H activity or AGO2, which are prevented due to the modifications on the protectors. RNase H degradation would happen if there was any DNA segment (unmodified at the 20 position) that could activate RNase H. LNAs

and 20 -O-methylated oligos cannot activate RNase H in the absence of DNA stretches (unmodified at the 20 position) in the oligo sequence as described [64,65,74,75] and therefore, do not cause mRNA degradation. Since only the specific target site is affected, while other AGO functions continue to be observed [49,69], these protectors likely do not interfere with AGO. This may be due to the fact that they are longer (27 nts or more) than the average microRNA/siRNA (19–24 nt) that binds AGO [34,35,38] and the 20 -O-methyl modification throughout the protector [49] can impair interaction with the RNAi machinery [37,65]. These features may ensure that the protectors affect only the specific complementary target site and do not associate or interfere with AGO complexes The oligos were synthesized commercially (IDT biotechnologies, IA). 3.3. Protocol B In our experiments, nucleofection is the best transfection method for our protector oligonucleotides as also suggested by antisense techniques [72,73]. The cells are grown and transfected as described above in protocol (A) (Fig. 1A) except that instead of siRNAs, 30–150 nM of the protector oligonucleotide is co-transfected with the reporters to block microRNA targets. A mutant version of the protector with impaired base-pairing ability (where one half of the 20 -O-methylated protector is mutated in two–three groups of mutations at different sections including the complementary sequence of the target microRNA base-pairing site) is tested in parallel. The mutant fails to bind the target and cannot prevent microRNA-target base-pairing, serving as a negative control. 3.4. Controls (1) Scrambled oligonucleotides that bear the same modifications as well as a buffer alone (the buffer in which the oligos were resuspended in, annealing buffer in our experiments) control are used in parallel experiments to modulate and ensure that antisense levels do not cause off-target effects. The scrambled sequence oligo acts as a negative control to normalize for

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Fig. 3. Protocol C. Disruption of microRNA-target mRNA base-pairing via mutational alterations of the microRNA target site on the mRNA, followed by complementation analysis of loss of function with compensatorily mutated microRNAs Direct microRNA-target mRNA interaction at a candidate target site required for target gene expression regulation can be validated by initially disrupting microRNA interactions and thereby, causing loss of function using mutational alterations to the target site in a 30 -UTR/target site-bearing reporter assay. The loss of microRNA levels and thereby, function is subsequently restored by complementing the mutant target with compensatorily mutated microRNA [11,23]. The consequent rescue of base-pairing of the compensatory microRNAs with the mutated sites restores microRNA-mediated gene regulation. (a) Basepairing of the target sites on the TNFa ARE reporter with miR369-3p microRNA leads to regulated gene expression of the reporter at the two specific target sites shown (orange). (b) In the case of the TNFa ARE reporter, mutating either one of the two target sites (shown in blue) or both sites (not shown) abrogates gene expression regulation, potentially due to loss of direct base-pairing interactions with the microRNA, miR369-3p. (c) The loss of base-pairing of miR369-3p microRNA with mutated target sites on the TNFa ARE reporter can be rescued by complementation with compensatorily mutated miR369-3p microRNA (miRseedmt369-3p, blue microRNAs). The restored productive base-pairing of the compensatory microRNAs with the mutated target sites should rescue gene expression regulation if direct microRNA base-pairing at these target sites is required to elicit microRNA-regulated gene expression of the TNFa ARE reporter.

non-specific effects of the modifications. The buffer-only transfected control enables controlling for effects of modified oligo addition. (2) In parallel with these experiments, a mutated version of the protector that fails to bind and cannot preclude microRNAtarget base-pairing can be tested as a negative control, as described above. (3) To validate that the loss of microRNP function observed was due to loss of base-pairing via interference at the target site by the LNA/20 -O-methyl protector and not due to non-specific effects, decoys (stable LNA/20 -O-methyl modified antisense to the protector sequences, excluding the complementary sequence of the target microRNA base-pairing site on the protector, to prevent interference with the microRNA) are used. The decoys mimic the sequences on the target mRNA that are adjacent to the microRNA base-pairing site but do not include the microRNA base-pairing site itself to prevent interference with the microRNA. The decoys can bind the protector with high affinities (due to the LNA/20 -O-methyl modification) and are transfected in parallel control experiments, at higher concentrations to that of the protector (60–300 nM) to titrate away the interfering target protector away from the target site and restore microRNA-mediated expression. (4) The target mRNA is expressed as a cDNA (without its 30 -UTR or with a control polylinker 30 -UTR lacking the sequences targeted by the protector) to rescue expression in the case of microRNA-mediated activation [49]. (5) AGO2/microRNP purification using anti-AGO2 antibody (cat#07-590, Millipore or clone 4G8 antibody from Wako) can be performed to immunoprecipitate AGO2-associated targets, followed by RT-PCR analyses to ensure restoration of the target mRNA onto AGO complexes coincident with recovery of microRNA-mediated target gene expression upon addition of decoys that sequester the target protector. (6) The target protector will also affect the endogenous target mRNA expression, which can be analyzed by Western blotting in parallel with the reporter assay and should show a similar response [49].

3.5. Potential issues (1) Excess amounts of modified oligos may show toxicity/ off-target effects and must be titrated and controlled for with a negative control bearing the same number and position of modifications as well as a buffer control. (2) In our experiments, nucleofection appeared efficient compared to other transfection methods as also observed with the use of 20 -O-methylated protectors in antisense techniques [72,73]. Both LNAs and 20 -O-methyl protectors were functionally effective in mammalian cells and in oocytes, where they were injected at the animal pole/nucleus [49]. (3) Blockage of the microRNA target site may in some cases, stabilize the mRNA and increase its expression. While this would be consistent with loss of function in the case of microRNA-mediated repression, it would not cause a loss of function effect in the case of microRNA-mediated activation. This can be checked by controls with decoys or mutated control protectors and additionally, a parallel experiment with antagomirs (Fig. 2c) to block the microRNA and cause a loss of function/activation. 4. Disruption of microRNA-target mRNA base-pairing via mutational alterations of the microRNA target site on the mRNA, followed by complementation analysis of loss of function with compensatorily mutated microRNAs (Protocol C) 4.1. Approach This method validates direct microRNA-target mRNA interaction at a candidate target site by initially disrupting microRNA interactions, using mutational alterations to the target site, and thereby, causing loss of function in a 30 -UTR/target site-bearing reporter assay (Fig. 3a and b). The loss of target-microRNA base-pairing and thereby, function is subsequently restored by

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complementing the mutant target with compensatorily mutated microRNA [11,23] (Fig. 3c). The requirement for direct association of the microRNA with the target RNA for regulated target expression can be distinguished from the role of UTR/target site secondary structure or of UTR/target site-associated RNA binding proteins with addback of compensatorily mutated microRNAs that should rescue base-pairing with the mutated sites. The consequent rescue of function in a dose dependent manner by the compensatorily mutated microRNA but not other control microRNAs added back, confirms the functional requirement for direct base-pairing between the microRNA and target mRNA for mediating microRNA functions (Fig. 3). Unlike the above two approaches, in this method, the endogenous microRNA and target gene expression are not affected and cannot be analyzed in conjunction with the reporter assay. 4.2. Design: mutating the target site in UTR reporters The target site is mutated within the microRNA seed complementary 2–8 nt region, usually at two or more nucleotides (the 2nd–5th sites). They are replaced by alternative nucleotides that would not basepair with the complementary sites on the corresponding microRNA (Fig. 3b). The resulting abrogated microRNA base-pairing causes loss of function in reporter assays. 4.3. Compensatory mutant microRNAs and controls The compensatory microRNAs are designed as siRNA duplexes or expressed from polymerase II driven vectors of pri-microRNAs encoding the duplex microRNA [40,41]. The compensatory microRNA bears mutations that would restore Watson–Crick base-pairing at the mutated target sites (Fig. 3c). The parent, non-mutated microRNA that cannot base-pair with the mutated target reporter as well as a scrambled microRNA control are also designed similarly and expressed as controls. The RNA oligos are either synthesized as individual sense and antisense, 50 -end phosphorylated (on the microRNA strand) RNA oligos (IDT biotechnologies, IA) and then annealed (protocol (A), annealing of RNA oligos) or as 50 phosphorylated duplexes (protocol (A)). Pri-microRNA expressing vectors that produce mature microRNAs in vivo, can be stably expressed for extended durations and can be induced/switched off using a Tet-on operator tetracycline/doxycycline responsive CMV promoter system as discussed in protocol (A) [41] (Open Biosystems).

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mutated microRNA. (2) Careful titrations are required to ensure that the mutant microRNAs are not over or under expressed leading to additional spurious effects or lack of rescue, respectively [11,43]. 4.6. Potential issues (1) Effects due to overexpression should be eliminated by titrating in the microRNA or titrating doxycycline for induction of the pri-microRNA inducible vectors, ensuring that levels of the exogenously-expressed, compensatory microRNA is comparable to that of the endogenous microRNA. (2) Any off-target effects should be eliminated by altering the sequence of the mutation and compensatory microRNA and by titrating to concentrations at which a scrambled control does not cause any effect. (3) The compensatory microRNA may be unstable and its levels must be induced and monitored carefully to ensure equal expression to that of the endogenous microRNA. The small RNA sequence can be altered with modified nucleotides (such as 20 -fluoro modified nucleotides) to impart more stability [37,64,65], used at higher levels or expressed from inducible vectors to obtain levels equivalent to the endogenous, natural microRNA. (4) The ratio of the mutant reporter to the compensatory microRNA is important to be maintained, where the compensatory microRNA levels are equimolar or higher [3]. Additionally, increasing microRNA levels excessively (higher than 200 nM) [38] is refractory because of the limiting unbound AGO available in the cell to form functional microRNPs with the exogenous, compensatory microRNA [42]. (5) Mutations in the target site may alter the structure or create a new binding site for RNAs or RNA-binding proteins and the compensatory microRNA may not be able to access the site. Alternative bases should be mutated to find sites that will elicit loss of function due to loss of basepairing and remain accessible to base-pairing with compensatory microRNAs. (6) The mutant target reporter may have reduced mRNA stability and the levels may decrease; the mutant reporter may need to be induced more than the non-mutated reporter to ensure expression equivalent to the non-mutated reporter for analysis. (7) The target reporter and the compensatory microRNA may be aberrantly localized due to the mutations. Localization should be monitored by fluorescence in situ hybridization studies or fractionation analyses. Alternative bases should be mutated to ensure that the mutant target reporter and compensatory microRNA localization follows that of the non-mutated reporter and endogenous, natural microRNA.

4.4. Protocol Nucleofection or Trans-it 293 transfection reagent (MIRUSBIO) methods were used according to the manufacturer’s instructions with the following modifications. The cells were grown and transfected as described above in protocol (A) (Fig. 1A) except that 50–100 nM of the mutant or control compensatory microRNAs were co-transfected with the control, wildtype and mutant target site reporters in parallel experiments. Alternatively, 250–500 ng of the vector that expresses the compensatory microRNA [41] was transfected as described above in protocol (A) (Fig. 1A) and doxycycline was titrated to find the right levels of induction of the microRNA (50–100 ng/ml in our experiments) [11,43]. 4.5. Controls (1) In parallel with these experiments, AGO2/microRNP purification using anti-AGO2 antibody (cat#07-590, Millipore or clone 4G8 antibody from Wako) can be performed to immunoprecipitate AGO2-associated targets, followed by RT-PCR analyses to ensure restoration of the mutated target mRNA on AGO2 complexes upon rescue of base-pairing with addback of the compensatorily

5. Materials HEK293 cell line (ATCC). tissue culture dishes and glassware and other tissue culture requirements. DMEM, FBS, L-glutamine, Penicillin/Streptomycin, trypsin (Invitrogen). Propidium Iodide (Sigma), ethanol, FACSCalibur Flow Cytometer (BD Biosciences). Nucleofection reagents for the cell line as per manufacturer’s instructions and nucleofector (Amaxa/Lonza). Trans-it 293 transfection reagent (MIRUSBIO). Firefly and Renilla Luciferase reporters [11,20,43]. pLVX-tet-on advanced plasmid (Clontech). pTRIPZ tetracycline inducible system for shRNAs and the firefly reporter (Open Biosystems-Thermo Scientific). Doxycycline (Sigma). siRNAs, microRNAs as duplexes and LNAs/20 -O-methyl oligos (IDT DNA Technologies).

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Antibodies: anti-AGO2, cat#07-590, Millipore or anti-AGO2 clone 4G8 antibody from Wako. Dual Luciferase assay system (Promega) and TD2020 luminometer (Turner Biosystems). RT-PCR supplies from Invitrogen and any RT-PCR machine (LightCyclerÒ 480 Real-Time PCR System) and 96–384 wells plates (Roche). 6. Summary These methods will confirm the functional requirement of direct interactions between microRNAs and their target RNAs. Similar assays can also be conducted for target RNAs that are not mRNAs, analyzing the functional requirement of microRNA interaction by assaying target RNA levels and functions [19] instead of mRNA UTR-regulated gene expression. A precise understanding of microRNA interactions with their targets as well as the development of analytical tools and associated interference techniques, such as target protectors [69], antagomirs [67] and RNA sponges [68], can lead to the emergence of potential, therapeutic approaches to manipulate specific gene expression and their consequent biological outcomes. Acknowledgments This work was supported by a Leukemia and Lymphoma Society Special Fellowship 3300-09, a Cancer Research Institute Investigator, the D. and M-E Ryder, Smith Family Foundation Awards and MGH start-up funds to SV. I thank S. S. Truesdell and J. C. Schroeder for technical help and S. Lee and S.I. Bukhari for critical reading. References [1] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, R. Parker, Genes Dev. 20 (2006) 515– 524. [2] T.A. Farazi, J.I. Spitzer, P. Morozov, T. Tuschl, J. Pathol. 223 (2011) 102–115. [3] S. Mukherji, M.S. Ebert, G.X. Zheng, J.S. Tsang, P.A. Sharp, Nat. Genet. 43 (2011) 854–859. [4] J. Liu, M.A. Carmell, F.V. Rivas, C.G. Marsden, J.M. Thomson, J.J. Song, S.M. Hammond, L. Joshua-Tor, G.J. Hannon, Science 305 (2004) 1437–1441. [5] L. Wu, J. Fan, J.G. Belasco, Curr. Biol. 18 (2008) 1327–1332. [6] M.R. Fabian, T.R. Sundermeier, N. Sonenberg, Prog. Mol. Subcell. Biol. 50 (2010) 1–20. [7] Vasudevan S: Posttranscriptional Upregulation by MicroRNAs, Wiley Interdiscip. Rev. RNA, (2011), doi: 10.1002/wrna.121. [8] A.J. Giraldez, Y. Mishima, J. Rihel, R.J. Grocock, S. Van Dongen, K. Inoue, A.J. Enright, A.F. Schier, Science 312 (2006) 75–79. [9] L. Wu, J. Fan, J.G. Belasco, Proc. Natl. Acad. Sci. USA 103 (2006) 4034–4039. [10] H. Guo, N.T. Ingolia, J.S. Weissman, D.P. Bartel, Nature 466 (2010) 835–840. [11] S. Vasudevan, Y. Tong, J.A. Steitz, Science 318 (2007) 1931–1934. [12] M. Chekulaeva, W. Filipowicz, Curr. Opin. Cell Biol. 21 (2009) 452–460. [13] A. Aravin, T. Tuschl, FEBS Lett. 579 (2005) 5830–5840. [14] D.P. Bartel, Cell 136 (2009) 215–233. [15] R.W. Carthew, E.J. Sontheimer, Cell 136 (2009) 642–655. [16] H.R. Chiang, L.W. Schoenfeld, J.G. Ruby, V.C. Auyeung, N. Spies, D. Baek, W.K. Johnston, C. Russ, S. Luo, J.E. Babiarz, R. Blelloch, G.P. Schroth, C. Nusbaum, D.P. Bartel, Genes Dev. 24 (2010) 992–1009. [17] J. Ule, K.B. Jensen, M. Ruggiu, A. Mele, A. Ule, R.B. Darnell, Science 302 (2003) 1212–1215. [18] M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger, A. Rothballer, M. Ascano Jr., A.C. Jungkamp, M. Munschauer, A. Ulrich, G.S. Wardle, S. Dewell, M. Zavolan, T. Tuschl, Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP, Cell 141 (2010) 129–141. [19] D. Cazalla, T. Yario, J.A. Steitz, Science 328 (2010) 1563–1566. [20] S. Vasudevan, J.A. Steitz, Cell 128 (2007) 1105–1118. [21] U.A. Orom, F.C. Nielsen, A.H. Lund, Mol. Cell 30 (2008) 460–471. [22] A. Lal, M.P. Thomas, G. Altschuler, F. Navarro, E. O’Day, X.L. Li, C. Concepcion, Y.C. Han, J. Thiery, D.K. Rajani, A. Deutsch, O. Hofmann, A. Ventura, W. Hide, J. Liebermanm, Capture of MicroRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling, PLoS Genet. 7 (2011) e1002363. [23] C.L. Jopling, M. Yi, A.M. Lancaster, S.M. Lemon, P. Sarnow, Science 309 (2005) 1577–1581.

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