[Methods in Molecular Biology] Polyadenylation Volume 1125 || RHAPA: A New Method to Quantify...

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Chapter 14

RHAPA: A New Method to Quantify Alternative Polyadenylation

Ashley L. Cornett and Carol S. Lutz

Abstract

3′ end formation of eukaryotic messenger RNAs (mRNAs) is an essential process that infl uences mRNA stability, turnover, and translation. Polyadenylation is the process by which mRNAs are cleaved at specifi c sites in response to specifi c RNA sequence elements and binding of trans-acting protein factors; these cleaved mRNAs subsequently acquire non-templated poly(A) tails at their 3′ ends. Alternative polyadenyl-ation occurs when multiple poly(A) signals are present in the primary mRNA transcript, in either the 3′ untranslated region (3′UTR) or other sites within the mRNA, resulting in multiple transcript variants of different lengths. We demonstrate here a new method, termed RHAPA ( R Nase H a lternative p olyadenyl-ation a ssay), that employs conventional RT–PCR with gene-specifi c oligonucleotide hybridization and RNase H cleavage to directly measure and quantify alternatively polyadenylated transcripts. This method gives an absolute quantifi ed expression level of each transcript variant and provides a way to examine poly(A) signal selection in different cell types and under different conditions. Ultimately, it can be used to further examine posttranscriptional regulation of gene expression.

Key words Polyadenylation , Alternative polyadenylation , 3′ end formation , RNase H , Oligonucleotide , 3′ untranslated region

1 Introduction

Polyadenylation is an essential processing event that forms the 3′ ends of all eukaryotic mRNAs, with the exception of histone mRNAs. The process of polyadenylation is accomplished by the actions of multi-subunit protein complexes which bind to specifi c RNA sequences in the 3′ untranslated region (3′UTR) of mRNAs. Alternative polyadenylation, that is, the presence and use of more than one polyadenylation signal, is now understood to occur in more than half of all human mRNAs [ 1 ]. Recent studies have high-lighted that alternative polyadenylation is an important regulatory mechanism of gene expression [ 2 – 7 ]. Alternative polyadenylation is important not only in defi ning the 3′UTR length but also in inclusion or exclusion of additional RNA elements, such as AU-rich stability elements or microRNA (miRNA) binding sites.

Joanna Rorbach and Agnieszka J. Bobrowicz (eds.), Polyadenylation: Methods and Protocols, Methods in Molecular Biology, vol. 1125, DOI 10.1007/978-1-62703-971-0_14, © Springer Science+Business Media New York 2014

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As shown in Fig. 1 , cleavage and subsequent poly(A) tail addition can result in RNAs of varying 3′UTR lengths. Polyadenylation at the 3′ most poly(A) signal (“distal” with respect to the stop codon) would result in an mRNA containing a 3′UTR of greatest length (shown in Fig. 1 as yellow), while polyadenylation at a more proxi-mal site would lead to a shortened 3′UTR (blue or green).

Analysis of alternative polyadenylation has been limited to two techniques until now: Northern blotting and RT–PCR. Northern blotting analysis requires use of a labeled probe to hybridize to a given complementary sequence within the RNA molecule of inter-est; however, it provides only a relative analysis of alternatively polyadenylated transcript expression. RT–PCR provides a more quantitative method of analyzing RNA expression; however, this quantitation is still a relative measure. Figure 2 depicts the mecha-nism of action of a standard RT–PCR reaction. In addition, RT–PCR does not account directly for use of the proximal polyadenylation signal as the primer set F1:R1 will not only amplify the short RNA transcript, but it will also amplify the long RNA transcript.

Although RT–PCR does not provide an absolute method to quantify alternatively polyadenylated transcripts, it was a starting point for our design of RHAPA, or R Nase H a lternative p olyade-nylation a ssay, as depicted in Fig. 3 . RHAPA employs the princi-ples of RT–PCR but requires the RNA to undergo hybridization with a gene-specifi c DNA oligonucleotide that will anneal to the RNA between the polyadenylation signals. RNase H treatment ensues, which will specifi cally cleave the DNA:RNA hybrid. Next, the cleaved RNA is converted into cDNA by reverse transcription (RT) with an oligo d(T) primer, which will only amplify transcripts that have a poly(A) tail. The cDNA is then used for quantitative PCR analysis.

AAAAAAAAAAn

AAAAAAAAAn

AAAAAAAAAAn

pA pA pA

Fig. 1 Schematic representation of alternative polyadenylation. In this example, multiple poly(A) signals are all located in the 3′-most exon. As a result, RNA transcripts generate 3′ untranslated regions (UTRs) of varying lengths. Exons are represented as red boxes ; introns are represented by black lines . Varying lengths of 3′ UTRs are represented by blue , green , and yellow boxes . pA, poly(A) signal


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Our new RHAPA method described here has several advan-tages. The method not only provides accurate and absolute quan-tifi cation of alternatively polyadenylated transcripts but is fast (~1–1.5 days) and reliable. It can be applied to any gene of interest and can be adapted to be used for multiple treatments or time- course experiments. It can also be easily coupled with global meth-ods such as RNASeq or microarray analysis to get a broad picture of gene expression regulation.

2 Materials

1. DMEM complete: Dulbecco’s Modifi ed Eagle’s Medium (DMEM) with 4,500 mg/mL glucose/L, L -glutamine, NaHCO 3 , pyridoxine HCl (Sigma Aldrich, St. Louis, MO, USA), 5 % fetal bovine serum (FBS,), 4 mM additional L -glutamine.

2. 10× phosphate-buffered saline (PBS), pH 7.2. 3. 1× PBS: Dilution of 100 mL of 10× PBS described above into

900 mL of sterile autoclaved dH 2 O. 4. Trypsin-ethylenediaminetetraacetic acid (EDTA) (1×).

2.1 Tissue Culture

STOP

Short (proximal) RNA Transcript

AAAAAAAAAA

Long (distal) RNA Transcript

AAAAAAAAAATTTTTTTTTTT

TTTTTTTTTTT

Short (proximal) cDNA

Long (distal) cDNA

RT-PCR (oligo d(T))

F1

R1

R2

F2F1

R1

pA pA

Fig. 2 Schematic of RT–PCR and problems with quantitative analysis. This diagram is a schematic representation of why standard RT–PCR is not suffi cient to quantify transcripts that result from alternative polyadenylation. A specifi c primer set (F1:R1) cannot be used to amplify the short RNA transcript alone, because those primers will also cause amplifi cation of the long mRNA. The short RNA transcript refers to mRNA polyadenylated at the proximal poly(A) signal, with respect to the stop codon; the long RNA transcript refers to mRNA polyadenylated at the distal poly(A) signal


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1. 1× PBS. 2. TRIzol reagent (Invitrogen Corp.). 3. Chloroform. 4. Isopropanol. 5. 80 % Ethanol: To make 500 mL, dilute 400 mL of 200-proof

absolute, anhydrous ethyl alcohol in 100 mL of sterile RNase- free double-distilled H 2 O.

6. Sterile RNase-free double-distilled H 2 O: Place double-distilled water in RNAse-free containers that have been used only for RNase-free double-distilled H 2 O. Autoclave the bottles fi lled with double-distilled H 2 O to sterilize them.

1. 1× DNA oligonucleotide annealing buffer: 10 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, pH 7.5–8.0.

2. 500 ng/μL gene-specifi c antisense DNA oligonucleotide ( see Note 1 ).

2.2 Total RNA Isolation

2.3 Gene-Specifi c Oligonucleotide Hybridization and RNase H Cleavage

Hybridize total RNA with DNAantisense oligonucleotides

AAAAAnAUUAAA AAAAAn

AAAAAAnAAAAAn

AAAAAn

AAAAAn

RNase H Cleavage

TTTTTn

TTTTTn

Oligo d(T)

Two Alternatively Polyadenylated Transcripts

AUUAAA

AUUAAA

AAUAAA

AAUAAA

AUUAAA

AAUAAA AAUAAA

AUUAAA

AAUAAA

AAAAAn

RT-PCR

XOligo d(T)

RT-PCR

Short (proximal) mRNA levels Long (distal) mRNA levels

AAUAAA TTTTTTTTTF1

R1AAUAAA TTTTTTTTT

F2

R2

Fig. 3 RHAPA mechanism of action. This assay is designed as an answer to accurate and faithful quantifi cation of alternatively polyadenylated transcripts. This method employs RNase H digestion of RNA:DNA hybrids to cleave RNA sequences between two given polyadenylation signals, which allow the separate quantifi cation of alternatively polyadenylated transcripts. The fi rst step involves the hybridization of the DNA oligonucleotide to its complimentary RNA sequence, followed by subsequent RNase H treatment. The 3′ cleaved RNA is then converted into cDNA by reverse transcription (RT) with an oligo d(T) primer, thereby only amplifying transcripts with a poly(A) tail. The cDNA is then used for PCR analysis. Poly(A) signals are shown in blue (proximal) and yellow (distal)


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3. RNase H (Invitrogen Corp). 4. Heat block: set at 70 °C.

1. ThermoScript RT (15 U/μL) (Invitrogen Corp.). 2. 5× cDNA synthesis buffer (Invitrogen Corp.). 3. 50 μM oligo (dT) 20 . 4. 0.1 M DTT (Invitrogen Corp.). 5. RNaseIN RNase inhibitor (Promega Corp., Madison, WI, USA). 6. Sterile RNase-free double-distilled H 2 O. 7. Template RNA (500 ng–1 μg/reaction). 8. 10 mM 2′deoxynucleoside-5′-triphosphate (dNTP): Dilute

10 μL 100 mM deoxycytidine triphosphate (dCTP), 10 μL of 100 mM deoxyadenosine triphosphate (dATP), 10 μL of 100 mM deoxyguanosine triphosphate (dGTP), and 10 μL of 100 mM thymidine triphosphate (dTTP) in 60 μL distilled dH 2 O.

1. 10× PCR buffer: 200 mM Tris–HCl (pH 8.4), 500 mM KCl. 2. 50 mM MgCl 2 . 3. Taq polymerase (5 U/μL). 4. 10 mM dNTP. 5. cDNA from fi rst-strand (RT) reaction. 6. 10 μM gene-specifi c isoform 1 forward primer (F1). 7. 10 μM gene-specifi c isoform 1 reverse primer (R1). 8. 10 μM gene-specifi c isoform 2 forward primer (F2). 9. 10 μM gene-specifi c isoform 2 reverse primer (R2). 10. 10 μM gene-specifi c control for cleavage forward primer (F3). 11. 10 μM gene-specifi c control for cleavage reverse primer (R3). 12. 10 μM housekeeping gene forward primer (such as primer spe-

cifi c for GAPDH). 13. 10 μM housekeeping gene reverse primer (such as primer spe-

cifi c for GAPDH). 14. PCR Cycler: PTC-100, Programmable Thermal Controller

(MJ Research™ Inc., Boston, MA, USA).

1. Sterile RNase-free double-distilled dH 2 O. 2. Agarose (Invitrogen Corp.). 3. 10× Tris–Borate–EDTA buffer (TBE): 1 M Tris, 0.9 M boric

acid, and 0.01 M EDTA. 4. 1× TBE buffer: Dilute 100 mL of 10× TBE buffer into 900 mL

sterile RNase-free double-distilled H 2 O.

2.4 Reverse Transcription (RT)

2.5 Polymerase Chain Reaction (PCR)

2.6 Agarose Gel Electrophoresis Components


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5. Ethidium bromide. 6. DNA gel loading dye: 15 % glycerol, 0.05 % w/v bromophe-

nol blue, 0.05 % w/v xylene cyanol. 7. DNA 100 bp ladder (Invitrogen). 8. Gel box. 9. Combs.

1. GeneGenius bio-imaging system (Syngene, Frederick, MD, USA).

2. GeneSnap image acquisition software (Syngene). 3. GeneTools analysis software (Syngene).

3 Methods

Use of the TRIzol method as per the manufacturer’s protocol for RNA extraction is provided below. However, other RNA isolation techniques can be employed.

1. Cells are grown to 80 % confl uency in DMEM + 5 % FBS. 2. Media is removed and cells are then washed with 1× PBS. 3. TRIzol reagent (1 mL) is added to each 60 mm dish and left

on the cells for 5 min at room temperature, allowing cell lysis ( see Note 3 ).

4. Cell mixture is added to 1.5 mL microfuge tubes for further steps.

5. 200 μL chloroform is added (1/5 of the original volume of TRIzol used) to tubes. Tightly secure microfuge tube caps, and vigorously shake by hand for 15 s. The mixed solution is then incubated at room temperature for 10 min followed by centrifugation (~10,000 × g ) in a microcentrifuge for 15 min.

6. After centrifugation, the resulting mixture separates into a lower, red phenol-chloroform phase, an interphase, and a col-orless upper aqueous phase. The RNA-containing aqueous phase is removed and transferred to a fresh microfuge tube.

7. Precipitate the RNA from the aqueous phase by mixing with isopropanol. Use 0.5 mL isopropanol for every 1 mL of TRIzol reagent used at the initial step. Incubate for 10 min at room temperature and centrifuge for 10 min at 10,000 × g . RNA will precipitate into a pellet at bottom of the tube. Gently decant supernatant to avoid disrupting the RNA pellet.

8. To wash the RNA pellet, add an equal volume (1 mL) of 80 % ethanol. Mix the sample gently and centrifuge at 7,500 × g for 5 min at 2–8 °C.

2.7 Detection and Analysis Equipment and Software ( See Note 2 )

3.1 RNA Isolation


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9. Briefl y dry RNA pellet, and resuspend in 60 μL RNase-free H 2 O. 10. Store RNA at −80 °C.

1. Incubate 1.5 μg template RNA with 2 μg of gene-specifi c DNA oligo in 50 μL 1× oligonucleotide annealing buffer in a microfuge tube.

2. Mix briefl y, then centrifuge for 15 s at 7,500 × g . 3. Place tube in a standard heat block set at 70 °C for 10 min,

then remove heat block from the apparatus, allowing tube and heat block to slowly cool to room temperature on the work bench ( see Note 4 ).

4. After the tube and mixture therein cools to ~25 °C, add 1 μL (2 U/μL) of RNase H (Invitrogen) and 0.5 μL (40 U/μL) of RNaseIN (Promega). Mix briefl y, and centrifuge briefl y for 10 s to collect contents to the bottom of the tube.

5. Incubate at 30 °C for 60 min ( see Note 5 ).

RNA purifi cation is carried out by QIAGEN RNeasy RNA cleanup protocol, and those steps are briefl y provided below. Other meth-ods to purify RNA can be applied ( see Note 6 ).

1. Adjust the sample volume to 100 μL with RNase-free water. Add 350 μL buffer RLT and mix.

2. Add 250 μL ethanol (100 %) to the dilute RNA, and mix by pipetting.

3. Transfer the sample to an RNeasy mini spin column placed in a 2 mL collection tube (supplied by the QIAGEN kit). Close lid, and centrifuge for 15 s at ≥8,000 × g . Discard fl ow through ( see Note 7 ).

4. Add 500 μL buffer RPE to the RNeasy spin column. Close lid, and centrifuge for 15 s at ≥8,000 × g to wash the spin column membrane. Discard fl ow through.

5. Add 500 μL buffer RPE to the RNeasy spin column. Close lid, and centrifuge for 2 min at ≥8,000 × g to wash the spin column membrane. Discard fl ow through.

6. Place RNeasy spin column in a new 1.5 mL collection tube (supplied by QIAGEN kit). Add 12 μL RNase-free water directly to the spin column membrane. Close the lid, and centrifuge for 1 min at ≥8,000 × g to elute the RNA.

7. Keep eluted RNA sample on ice and immediately proceed to Subheading 3.4 ( see Note 8 ).

Complimentary DNA (cDNA) is produced following the Invitrogen ThermoScript Reverse Transcriptase (RT) protocol. PCR is carried out by the Invitrogen Taq polymerase as per the manufacturers’ protocol.

3.2 Gene-Specifi c DNA Oligonucleotide Hybridization and RNA:DNA Hybrid Cleavage

3.3 RNA Purifi cation

3.4 Synthesis of cDNA and Polymerase Chain Reaction (RT–PCR)


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1. Incubate ~1 μg RNA (from Subheading 3.3 ), 1 μL 50 μM oligo(dT) 20 , 2 μL 10 mM dNTP mix in 12 μL reaction, and add RNase-free H 2 O to reach volume. Incubate mixture at 65 °C for 5 min.

2. To reaction mixture, add 4 μL 5× cDNA synthesis buffer, 1 μL 0.1 M DTT, 1 μL RNaseIN, 1 μL sterile distilled water, and 1 μL Thermoscript RT (15 U/μL). Mix gently and incubate at 50 °C for 60 min. Terminate reaction by heating at 85 °C for 5 min.

3. Proceed to step 4 or cDNA can be stored at 4 °C−20 °C. 4. Incubate 5 μL of 10× PCR buffer, 1.5 μL 50 mM MgCl 2 , 1 μL

10 mM dNTP mix, 1 μL of a sense and antisense GAPDH primer, or other housekeeping gene primer set, 1 μL of a sense and antisense gene of interest primer, and 0.5 μL Taq polymerase (1 U/1 μL). Dilute reaction to 50 μL in RNase-free H 2 O.

Step 1: Incubate PCR reaction at 95 °C for 5 min. Step 2: Denature 94 °C for 30 s. Step 3: Anneal 55 °C for 1 min. Step 4: Extend 72 °C for 30 s.

Repeat steps 2 –4 for a total of 30 cycles.

Step 5: Final extension 72 °C for 5 min. Step 6: Keep reaction at 4 °C after cycling.

Figure 4 diagrams the primer sets for the gene of interest and also how these primers are used to ensure RNAse H digestion ( see Note 9 ).

1. Prepare 1–1.5 % agarose gel. Add 1–1.5 g agarose to 90 mL of H 2 O in a 250 mL Erlenmeyer fl ask. Microwave agarose mix-ture until agarose dissolves in water ( see Note 10 ).

2. Allow to cool briefl y. Add 10 mL 10× TBE to fl ask. 3. Then add 10 μL ethidium bromide and immediately pour gel

into gel apparatus ( see Note 11 ). 4. While gel is solidifying, add 5 μL of 6× DNA gel loading dye

to each PCR reaction. 5. Each PCR reaction is subsequently separated by electrophore-

sis on a 1.5 % agarose gel for approximately 2 h at 100 V.

1. The agarose gel is placed on a gel imaging software docking station.

2. Bands are quantifi ed using GeneSnap tool. 3. Bands are normalized to the GAPDH mRNA control and are

expressed as a percent of total ( see Note 12 ). Figure 4 depicts an example of a gel revealing the results of RHAPA analysis.

3.4.1 PCR Cycling Conditions

3.5 Agarose Gel Electrophoresis

3.6 Analysis and Quantifi cation of Data


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4 Notes

1. Design the gene-specifi c antisense oligonucleotide under similar conditions as considered when designing a PCR primer: (a) The oligonucleotide should be roughly 18–22 nucleotides

in length. (b) The GC content should be roughly 40–60 %. (c) The melting temperature ( T m ) should be approximately

60 °C.

F1

R1

AAAAAAAA

Site of RHAPA cleavageR2

F2

Control for Cleavage

R3

F3

AAAAAAAA

AAAAAAAA

AAAAAAAA

GAPDH

CTRL for Cleavage

COX-2 Short RNA

a

b

COX-2 Long RNA

F1:R1

F2:R2

F3:R3

Fig. 4 RHAPA analysis. ( a ) Diagram depicting primer location and how the assay works. Blue pA, proximal polyadenylation signal; Yellow pA, distal polyadenyl-ation signal; and red box , site of DNA oligonucleotide annealing. The primer set F1:R1 will amplify a short, or proximal, RNA transcript. The primer set F2:R2 will amplify a long, or distal, RNA transcript. The primer set F3:R3 will test for com-pleteness of the RNase H cleavage reaction. Amplifi cation of a PCR fragment resulting from F3:R3 would indicate that the RNase H cleavage reaction did not occur to completion, and therefore, the quantifi cation would not be absolute. ( b ) Total RNA was isolated from Beas2B (normal lung cell line) and A549 (lung cancer) cells. RNA was subjected to COX-2-specifi c RHAPA analysis. Shown here are lanes from a representative ethidium bromide stained agarose gel


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2. Gel imaging software. 3. The amount of TRIzol reagent added is based on the area of

the culture dish (1 mL/10 cm 2 ). 4. Cooling to room temperature on a work bench takes roughly

45 min–1 h. 5. RNase H has enzymatic activity up to 37 °C. Heat blocks set

to a temperature in the range of 30–37 °C are appropriate. Do not exceed 37 °C.

6. Other methods to clean RNA can be employed; however, the RNeasy Kit: RNA cleanup (Qiagen) was selected based on the principle of purpose for this protocol. This method is designed to clean up RNA previously isolated by different methods and/or after enzymatic reactions, e.g., RNase H digestion. It also isolates RNA that is of a size larger than 200 nt; therefore, any excess DNA oligonucleotide in the reaction will remain in the QIAGEN column.

7. In depth description of protocol specifi cation can be found in the RNeasy Mini Handbook. The protocol can be downloaded at www.qiagen.com/literature/protocols/RNeasyMini.aspx .

8. After carrying out the RNA cleanup protocol, it can be assumed that residual starting template RNA is not eluted through each treatment; therefore, as a fail-safe, the RHAPA method requires 1.5 μg starting template RNA to compensate for RNA lost from step to step. With this assumption, at the point of the reverse transcription step, 1 μg of RNA is assumed to remain to serve as the template for cDNA.

9. PCR cycling conditions are based on melting temperature of the primer sets. The annealing temperature should be roughly 5 °C lower than melting temperature for the given primer. Also the extension time is based on the size of the PCR frag-ment, e.g., for every 1 kb the extension time should be 1 min.

10. Plug the 250 mL Erlenmeyer fl ask with a paper towel or tissue to avoid bubbling over of the agarose mixture.

11. Ethidium bromide (EtBr) is an intercalating hazardous agent. This material should be handled according to the material safety data sheet (MSDS).

12. In order to quantify PCR fragment bands, GeneTools has a “manual band quantifi cation” option. By manually drawing rectangular boxes around each band and setting each box to be the same size, this provides a way to quantify intensity of each band, using the intensity of the GAPDH PCR fragment band to normalize expression of the gene of interest.


http://www.qiagen.com/literature/protocols/RNeasyMini.aspx

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Acknowledgements

The authors wish to thank Anita Antes for careful reading of the manuscript. The authors also wish to thank the Lung Cancer Research Foundation and the American Heart Association, #12GRNT9120029 for grants awarded to C.S.L. Cheers to all.

References

1. Tian B, Hu J, Zhang H et al (2005) A large- scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33:201–212

2. Lutz CS, Moreira AM (2011) Alternative poly-adenylation of eukaryotic mRNAs: an effective regulator of gene expression. WIREs RNA 2:22–31

3. di Giammartino DC, Nishida K, Manley JL (2011) Mechanisms and consequences of alter-native polyadenylation. Mol Cell 43:853–866

4. Sandberg R, Neilson JR, Sarma A et al (2008) Proliferating cells express mRNAs with short-ened 3′ untranslated regions and fewer

microRNA target sites. Science 320:1643–1647

5. Mayr C, Bartel DP (2009) Widespread short-ening of 3′ UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138:673–684

6. Ji Z, Lee JY, Pan Z et al (2009) Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc Natl Acad Sci U S A 106:7028–7033

7. Shi Y (2012) Alternative polyadenylation: new insights from global analyses. RNA 18:2105–2117


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