[Methods in Enzymology] RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways Volume 449 || Co-Immunoprecipitation Techniques for Assessing RNA–Protein

C H A P T E R F I F T E E N

Methods

ISSN 0

Departm

Co-Immunoprecipitation Techniques

for Assessing RNA–Protein

Interactions In Vivo

Nicholas K. Conrad

Contents

1. In

in

076

en

troduction

Enzymology, Volume 449 # 2008

-6879, DOI: 10.1016/S0076-6879(08)02415-4 All rig

t of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas,

Else

hts

US

318

2. In
Vivo Ultraviolet Cross-Linking 321
2
.1. M aterials and buffers 322
2
.2. P rocedure 323
2
.3. R esults 326
3. C
ell Mixing Experiment 327
3
3
.2. P rocedure 329
3
.3. R esults 331
4. R
NA Immunoprecipitation 331
4
4
.2. P rocedure 333
4
.3. R esults 335
5. D
iscussion 336
6. C
oncluding Remarks 338
Ackn
owledgments 339
Refe
rences 339
Abstract

From the moment a nascent transcript emerges from an RNA polymerase until

its ultimate destruction, an RNA is bound by proteins that govern its fate. Thus,

in order to understand posttranscriptional regulation of gene expression, it is

essential to ascertain which proteins bind a given RNA in vivo. This chapter

describes three immunoprecipitation-based assays designed to query the

in vivo makeup of RNA–protein complexes. Two of these, UV cross-linking and

RNA immunoprecipitation (RIP), include cross-linking steps that trap complexes

formed in vivo. A third, a cell mixing experiment, verifies that an interaction

vier Inc.

reserved.

A

317

318 Nicholas K. Conrad

occurs in vivo by controlling for RNA–protein association subsequent to cell

lysis. Using these protocols, this chapter presents evidence that the abundant

nuclear RNA-binding protein hnRNP C interacts with the Kaposi’s sarcoma-

associated herpesvirus polyadenylated nuclear RNA in vivo.

1. Introduction

The effect of RNA-binding proteins on posttranscriptional generegulation depends on the specific protein–RNA interaction and the cellu-lar environment. The most thoroughly examined RNA elementcontrolling RNA degradation is the AU-rich element (ARE) found in the30-UTR of posttranscriptionally regulated mRNAs (reviewed in Barreauet al., 2005; Brennan and Steitz, 2001; Dean et al., 2004). The stability ofARE-containing mRNAs is mediated by cell signaling pathways that regu-late the binding and activity of specific proteins that either stabilize ordestabilize the transcript. While ARE-containing mRNAs may representparticularly intricately regulated transcripts, the general idea that specificproteins bind an mRNA and regulate its turnover is likely the rule ratherthan the exception. Therefore, experimental methods that examine thein vivo composition of ribonucleoprotein particles (RNPs) are essential inefforts to gain an understanding of the mechanisms that control transcriptstability.

Candidate RNA-binding proteins that regulate RNA decay (or anyother process) are often identified using affinity chromatography(Karijolich et al., 2007; Penalva and Keene, 2004; Ulbrich et al., 1980),RNA tags (Hogg and Collins, 2007; Srisawat and Engelke, 2002), ultraviolet(UV) cross-linking (Reed and Chiara, 1999), and biochemical purification(Wilson and Brewer, 1999). In some cases, validation of the relevance ofthe interaction can be provided by correlating an in vitro (de-)stabilizingactivity with binding (Bergman et al., 2007; Garneau et al., 2007). Even so,it remains essential to verify that the candidate protein has similar bindingand (de-)stabilizing activities in vivo. In mammalian cells, overexpressionstudies with both wild-type and dominant-negative factors and siRNA-mediated knockdown approaches are commonly used to confirm effects ofproteins on RNA stability (e.g., Yamashita et al., 2005; see Chapter 9 byMatsuda, Sato, and Maquat in this volume). Artificial tethering of proteinsto RNA transcripts is also often used to validate RNA-binding proteinactivity in vivo (Coller and Wickens, 2002; Lykke-Andersen et al., 2000).This chapter focuses on techniques used to confirm that a candidateRNA-binding protein interacts with a specific RNA in vivo.

Coimmunoprecipitation (co-IP) is a powerful means of examiningprotein–protein interactions (Harlow and Lane, 1988). A protein of interest

Analysis of RNA–Protein Interactions In Vivo 319

is purified by passing cell lysate over an antibody immobilized on a resin andcopurifying proteins are identified by Western blotting or by mass spec-trometry. Protein–RNA interactions can also be analyzed using co-IPtechniques in which coimmunoprecipitating transcripts are determinedby Northern blotting, reverse transcription-polymerase chain reaction(RT-PCR), RNase protection assays, or end labeling with radioisotopes.These techniques can provide reliable information about the nature ofprotein–RNA complexes in vivo (e.g., Hosoda et al., 2006; Jonson et al.,2007). Indeed, the history of RNA biology has been influenced greatlyby co-IP assays using human autoimmune sera (Keene, 1996; Lerner andSteitz, 1979; Lerner et al., 1981). More recently, microarray technology hasallowed global examinations of transcripts that coimmunoprecipitate with aspecific protein (Tenenbaum et al., 2002). Thus, RNA co-IP experimentsprovide a powerful tool to examine RNA–protein interactions.

As with any experimental technique, RNA co-IP experiments are notwithout their limitations. One potential problem is that RNA–proteincomplexes may dissociate upon cell lysis, producing false negatives(Fig. 15.1). Abundant, nonspecific RNA-binding proteins may competesuccessfully with specific proteins for a ligand RNA in extract. In vivo, highlocal concentrations of specific RNAs and proteins may support low-affinity interactions that are lost in cell extract due to dilution. Therefore,in order to detect an RNA–protein complex, it may be essential to securethe interaction by covalently cross-linking the components. This assertionis illustrated in a recent publication that describes the identification ofnovel components of an ARE-containing mRNA complex whose

Lysis

Figure 15.1 RNA^protein complexes can be gained or lost in the cell extract.The diagram depicts the association of RNA (wavy lines) with proteins (geometricshapes) in a cell (left) and in extract after lysis (right). Because the protein^RNA inter-actions represented by red cubes and lines do not reassociate, the interactions in celllysate mirror those found in vivo. In contrast, the green cylinders represent predomi-nantly unbound nuclear proteins. Upon lysis, these proteins maintain interactions withtheir cognate RNAs, but the free molecules can now access high-affinity ligand RNAs(purple) that had been segregated from them in the cytoplasm of intact cells.The bind-ing of proteins represented by purple hexagonal prisms is lost due to a high off-rate andsubsequent dilution in extract.


copurification was dependent on formaldehyde cross-linking (Vasudevanand Steitz, 2007).

In addition to losing interactions in extract, experimental observationsdemonstrate that RNA–protein associations can be gained upon cell lysis(Fig. 15.1). For example, the HuR protein, which binds to ARE-containingmRNAs (Brennan and Steitz, 2001; Fan et al., 1997; Myer et al., 1997; Penget al., 1998), interacts with transcripts in cell lysates that were not bound byHuR prior to lysis (Mili and Steitz, 2004). While multiple factors may beresponsible for such false-positive results, one possibility is that the proteinand RNA being investigated are localized to different compartments in vivo.When released from these compartments by cell lysis, the components caninteract freely. This phenomenon has been demonstrated empirically in thecase of the NAF1 protein, a factor involved in small nucleolar ribonucleo-protein particle (snoRNPs) biogenesis (Kittur et al., 2006). In vivo, NAF1does not associate with mature snoRNPs due to its exclusion from nucleoliand Cajal bodies, but NAF1 binds mature snoRNPs once cells are lysed.Consequently, the observation that an RNA coimmunoprecipitates with aspecific protein cannot be taken as an absolute proof of in vivo associationbetween that protein and RNA.

With currently published data, whether protein–RNA reassortment incell lysate is a rare exception or a more frequent occurrence is impossible todetermine. However, it is clear that, in many cases, the interactionsobserved in co-IP experiments mirror those that occur in vivo (e.g.,Hosoda et al., 2006; Jonson et al., 2007). Even in cases in which proteinsreassort, the selection of a specific RNA in cell lysate, among the manyRNA species present, may be a valuable piece of data. For example, inworks demonstrating that HuR reassociates in extract (Mili and Steitz,2004), HuR bound the c-fos mRNA in lysate but not in vivo. Importantly,in other conditions, c-fos mRNA is a bona fide target of HuR (Fan et al.,1997; Myer et al., 1997; Peng et al., 1998). Therefore, even though theycannot be interpreted as proof of an in vivo association, traditional RNAco-IP experiments do indeed yield biologically relevant information.

This chapter describes three different protocols designed to confirm thata particular protein–RNA complex forms in vivo, including UV cross-linking, cell mixing, and RNA immunoprecipitation (RIP) procedures.In each case, advantages and disadvantages of the individual proceduresare discussed and a detailed protocol is given. For illustration, an interactionbetween the Kaposi’s sarcoma-associated herpesvirus (KSHV) PAN RNAand a Flag-tagged version of hnRNP C1 is provided. PAN RNA is anoncoding polyadenylated transcript of unknown function that accumulatesto high nuclear levels during viral lytic phase (Sun et al., 1996; Zhong et al.,1996). The high nuclear abundance is due, at least in part, to a cis-actingelement called the ENE, which stabilizes transcripts both in nuclear extractsand in cultured cells (Conrad et al., 2006, 2007). In nuclear extracts, the


ENE efficiently cross-links to both splice isoforms of hnRNP C (hnRNP C1and C2; N. K. Conrad and J. A. Steitz, unpublished observation), an abun-dant nuclear RNA-binding protein involved in many aspects ofRNA metabolism (Dreyfuss et al., 1993; Krecic and Swanson, 1999). Resultsobtained from the three techniques described here all support the conclusionthat hnRNP C and PAN RNA interact in vivo.

2. In Vivo Ultraviolet Cross-Linking

In the more than 25 years since its first description (Mayrand andPederson, 1981; Wagenmakers et al., 1980), in vivo UV cross-linking hasproven to be a powerful tool in the examination of RNA–protein interac-tions that occur in cells. The technique is based on the ability of UV light tocovalently cross-link protein to RNA in living cells, without requiringchemical agents that may perturb the RNA–protein interface. UV irradia-tion is a ‘‘zero length’’ cross-linker; that is, it creates RNA–protein cross-links at the point of interaction. Thus, a UV-induced cross-link can beinterpreted as a demonstration of a direct interaction between an RNA andprotein. In contrast, the results of cell mixing or RIP assays only support theconclusion that an RNA and protein are in the same complex in vivo.

Several photochemical mechanisms have been reported for protein–nucleic acid UV cross-linking (Meisenheimer and Koch, 1997; Williamsand Konigsberg, 1991). In the most common, photoexcitation of a nucleicacid base results in free radical formation, followed by hydrogen atomabstraction from a nearby amino acid and covalent bond formation by freeradical recombination. Theoretically, almost any amino acid is suitable forcross-linking, but a favorably positioned hydrogen atom is required(Williams and Konigsberg, 1991). Other proposed mechanisms involvefree radical formation of an aromatic amino acid side chain or nucleophilicattack by lysine (Meisenheimer and Koch, 1997). These mechanisms are, bydefinition, more restrictive with respect to the specific amino acid require-ments at the protein–RNA interface. In addition to potential amino acidbiases, there is a preference for uridine to be the cross-linked base (Williamsand Konigsberg, 1991). Thus, one drawback of UV cross-linking is that theRNA–protein interface must be such that it supports the chemistry of thephotocross-linking reaction. Another is that the efficiency of cross-linkingin vivo is often lower than that achieved using chemical cross-linkers such asformaldehyde. Therefore, it is likely that certain RNA–protein interactionsmay remain undetected using this assay, even though the interaction existsin vivo. Finally, UV-induced cell damage could lead to alterations in theRNA–protein complexes in the cross-linked cells.


This section describes a modified in vivoUV cross-linking protocol basedon the method of Cook et al. (2004). While the example tests binding oftransiently expressed Flag-hnRNP C1 to PAN RNA, an advantage of UVcross-linking is that it can be used to examine endogenous RNA–proteincomplexes. To do so, an antibody must be available to the protein of interestthat recognizes the antigen under the following stringent conditions. Also,an isotype-matched negative control antibody is necessary to replace thevector alone control.

2.1. Materials and buffers

1. Transfection reagents if necessary. Any general protocol should work.2. Phosphate-buffered saline, pH 7.2 (PBS; Sigma), chilled on ice.3. UV source (Spectroline XL-1500 with 254-nm wavelength bulbs)4. 15-ml conical tubes5. Benchtop centrifuge [Sorvall Legend RTþ (Thermo-Fisher)]6. Microfuge tubes7. Room temperature and 4 �C microfuges8. Vortex9. Dry ice10. Phenol:chloroform:isoamyl alcohol, 25:24:1 (pH 7.5; PCA)11. Competitor RNA: a final concentration of 2.5 mg/ml of torula yeast

RNA (Sigma) and 0.5 mg/ml of polyuridylic acid potassium salt (poly-U;Sigma). The latter is especially helpful in preventing reassociation ofhnRNP C, as it has a high affinity for U-rich RNA. Poly-U may beunnecessary for other RNA-binding proteins or may be substituted withan RNA that binds the protein of interest. To prepare both torula yeastRNA and poly-U stock solutions, resuspend the RNA in 0.3M sodiumacetate (pH 5.2) and extract with an equal volume of PCA. PrecipitateRNA from the aqueous phasewith 2 volumes of 100% ethanol, washwith70% ethanol, and resuspend in RNase-free water to 50 mg/ml for torulayeast RNA and 40 mg/ml for poly-U.

12. SDS lysis buffer [0.5% (w/v) sodium dodecyl sulfate (SDS), 50 mMTrisHCl (pH 6.8), 1 mM EDTA, 1 mM dithiothreitol (DTT), 10 mMvanadyl–ribonucleoside complex (VRC; New England Biolabs), 1 mMphenylmethylsulfonyl fluoride (PMSF), competitor RNA]. Make freshevery experiment; add PMSF immediately before use.

13. Water baths at 65 and 37 �C14. RIPA correction buffer [1.25% NP-40, 0.625% sodium deoxycholate,

62.5 mM Tris–HCl (pH 8.0), 2.25 mM EDTA, 187.5 mM sodiumchloride, 10 mM VRC, 1 mM PMSF, competitor RNA]. Make freshevery experiment; add PMSF immediately before use.

15. RIPA buffer [1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,150 mM sodium chloride, 50 mM Tris–HCl (pH 8.0), 2 mM EDTA].


16. Anti-Flag M2 affinity gel (Sigma), washed three times in RIPA buffer.Approximately 40 ml of slurry per sample is used, which corresponds to�20 ml bead volume. If using an antibody directed against an endoge-nous protein, it should be bound to beads using standard protocols(Harlow and Lane, 1988) and washed three times in RIPA buffer.

17. Nutator18. QIAshredder spin columns (Qiagen)19. Control RNA. This serves as a control for loading and recovery.

An in vitro-transcribed b-globin ‘‘sense’’ RNA is used here, as it is notexpressed in HEK 293 cells and its size does not overlap that of PANRNA on a Northern blot.

20. Proteinase K solution [0.5 mg/ml proteinase K, 0.5% SDS, 20 mMTris–HCl (pH 7.5), 5 mM EDTA, 16.7 ng/ml GlycoBlue (Ambion),0.1 mg/ml torula yeast RNA, 6.7 pg/ml control RNA].

21. 3 M sodium acetate (pH 5.2)22. 100% ethanol23. Rubber policeman

2.2. Procedure

1. Transfect cells. In a minimal experiment, three transfections are neces-sary. Transfect two 10-cm plates with both protein and ligand RNAexpression constructs: one plate will be the cross-linked ‘‘test’’ samplewhile the other serves as the ‘‘no UV’’ control. Transfect a third platewith the RNA expression construct but no protein expression con-struct. This negative control verifies that any binding observed isnot a consequence of nonspecific binding to the antibody beads. Ifperforming an experiment assaying an endogenous protein–RNAinteraction, one plate is cross-linked, one plate is a ‘‘no UV’’ control,and the third is a cross-linked plate that will be immunoprecipitatedwith a control antibody (isotype matched to the protein-specific anti-body). For the experiment shown in Fig. 15.2, TransIT-293 (Mirus)was used to transfect each of two 10-cm plates with 4 mg of PAN-WT(Conrad and Steitz, 2005), 4 mg of pRTS-ORF50 [this plasmid encodesa viral factor necessary for PAN RNA transcription (Chang et al., 2002;Song et al., 2002)], and 4 mg of Flag-hnRNP C1 expression construct.The negative control plate was transfected with PAN-WT, pRTS-ORF50, and pcDNA3 (Invitrogen).

2. Approximately 18–24 h posttransfection, wash the plates with 5 ml ofice-cold PBS. Avoid detaching cells from plate. Plates should be�70 to100% confluent, corresponding to �107 cells.

3. Add 3 ml of ice-cold PBS. Keep dishes level on ice, making sure toavoid dry areas on the plate.

UVFlag-C1 + −+ + −+ + −+

− ++ − ++ − ++

Input Supe Pellet

PAN

Control

7SK

0.6 0.724

Flag-C1

1 2 3 4 5 6 7 8 9

% PAN IP

Figure 15.2 PAN RNA efficiently cross-links hnRNP C in transiently transfectedHEK293 cells. (Top) Northern blot analysis of a representative in vivoUVcross-linkingexperiment.Whether samples were transfected with Flag-hnRNP C1 and/or exposedtoUVirradiation is indicated above each lane.The topband is PANRNAand thebottomband is exogenously added b-globin transcript used as a control for gel loading andRNA recovery. The input and supernatant lanes represent only 5% of the total whilethe pellet lanes are 100%. (Middle) The same blot was probed for 7SK RNA, an abun-dant nuclear transcript that has not been reported to bind hnRNPC.The percent PANRNA immunoprecipitated is shown beloweach of the pellet lanes on theNorthern blot.Quantitation was performed using a phosphorimager and ImageQuant analysis soft-ware (Amersham). Each of the input and pellet PAN RNA bands was ‘‘boxed’’and anequivalent background ‘‘box’’ was generated below the PAN RNA signal.The volumeof each background box was then subtracted from the PAN RNA signal; a similarbackground correction was performed for the b-globin bands. PAN RNAvalues werenormalized by dividing the background-corrected PAN RNA signal by thebackground-corrected b-globin signal.The normalized pellet valueswere subsequentlydivided by the normalized input value for the corresponding sample and multipliedby five to correct for the fraction of input loaded on the gel (i.e., lane 7 normalizedvalue/lane 1 normalized value � 5 ¼ % PAN IP). (Bottom) Western blot of proteinfractions from the same experiment probed with an anti-Flag polyclonal antibody(Sigma). Lanes are equivalent to the Northern blot, except the input and supernatantlanes contain10%of the total.


4. UV cross-link the ‘‘test’’ plate and the negative control plate. To cross-link, place the dishes (without covers) on ice �3 cm from the bulbs of aUV source. The samples shown in Fig. 15.2 were irradiated with250 mJ/cm2. However, it may be necessary to adjust this parameter;�125 to 1500 mJ/cm2 is a reasonable range to test. In general, longertimes result in increased RNA degradation, but a greater chance ofcross-linking the protein to RNA. Keep the ‘‘no UV’’ control cells onice while the other plates are being irradiated.

5. Add 7 ml of ice-cold PBS to each plate and harvest cells by scrapingwith a rubber policeman; transfer to 15-ml conical tubes.

6. Pellet cells by centrifuging at 700g for 3 min at 4 �C and remove PBS.7. Add 1 ml of ice-cold PBS and transfer to microfuge tubes.


8. Centrifuge at 2400g for 1 min at 4 �C.9. Remove PBS and snap freeze on dry ice. At this stage, pellets can be

stored indefinitely at –80 �C.10. Thaw pellets on wet ice and vortex briefly (�2 s).11. Add 140 ml of SDS lysis buffer. Lysis will liberate genomic DNA,

causing the solution to become very viscous. It is not necessary tofully homogenize the sample at this step. Mix as well as is reasonable,without losing sample.

12. Heat samples to 65 �C for 5 min. Higher temperatures may be necessaryto decrease the signal in the ‘‘no UV’’ control for particular proteins,but may lead to more RNA degradation.

13. Chill on ice for 2–3 min.14. Add 560 ml of ice-cold RIPA correction buffer.15. Pass solution through a QIAshredder spin column by centrifugation at

16,000g at 4 �C for 1 min. Repeat. This step shears DNA, therebyreducing viscosity dramatically and increasing RNA recovery. Sonica-tion can be substituted for the QIAshredder step, but may lead tosignificant increases in RNA degradation.

16. Centrifuge extract at 16,000g at 4 �C for 15 min. Repeat twice for atotal of three spins; pellet should be minimal after the third spin.

17. Remove 35 and 3.5 ml of the lysate and place on dry ice. These are the5% RNA and 10% protein ‘‘input’’ samples, for Northern and Westernblot analysis, respectively.

18. Add remaining lysate to washed antibody-bound beads and nutate at4 �C for 2 h.

19. Centrifuge at 845g at room temperature for 1 min.20. Remove 35 and 3.5 ml and place on dry ice. These are the 5%RNA and

10% protein ‘‘supernatant’’ samples for Northern and Western blotting,respectively.

21. Discard remaining supernatant.22. Add 500 ml of ice-cold RIPA to beads, centrifuge at 845g at room

temperature for 1 min, and discard supernatant. Repeat four times for atotal of five washes.

23. Resuspend beads in a total volume of 200 ml. Remove 10 ml of slurryand place on dry ice. This is the protein ‘‘pellet’’ sample for Westernblotting.

24. Centrifuge the remaining slurry at 845g at room temperature for 1 minand discard supernatant.

25. Thaw RNA ‘‘input’’ and ‘‘supernatant’’ samples at room temperature.Add 300 ml of proteinase K solution to input, supernatant, and beads.

26. Incubate at 37 �C for 1.5 h. It is important to fully degrade cross-linkedproteins or RNA will be lost in the organic phase during PCAextraction.


27. Add 30 ml of 3 M sodium acetate (pH 5.2), 350 ml PCA, vortex for 5–10 s, and centrifuge at 16,000g at room temperature for 5 min.

28. Transfer upper aqueous layer to a tube containing 900 ml of 100%ethanol. Ethanol mix can be stored indefinitely at –20 �C.

29. Analyze using standard Northern blot (Fig. 15.2, top) or RNase pro-tection assay protocols. About one-third to one-half of the ethanol mixis usually sufficient to observe a signal. In general, RT-PCR should notbe used to detect RNA. While the bulk of cross-linked protein isremoved by the proteinase K treatment, the covalent bonds are notreversed; therefore, small adducts persist that inhibit elongation byreverse transcriptase. Even with relatively small amplicons (�100 bp),significant decreases have been observed in RT-PCR amplification ofPAN RNA from UV-treated cells compared with untreated cells.In some cases, the region chosen for RT-PCR amplification may notsupport protein–RNA cross-links that would decrease RT efficiency.Thus, RT-PCR can be used as a detection method if the UV-treatedand untreated ‘‘input’’ samples show similar amplification efficiencies(Hosoda et al., 2006).

30. Protein samples should also be analyzed using Western blot protocols toverify efficient immunoprecipitation (IP) of the protein of interest(Figure 15.2, bottom). For data shown in Fig. 15.2, one-quarter ofthe saved fractions was used for Western analysis. Prior to Westernanalysis, the proteins in the pellet fractions must be stripped from thebeads by boiling for 3 min in standard SDS-PAGE loading buffer.

2.3. Results

Figure 15.2 shows the results of an in vivo UV cross-linking experimenttesting the proposed interaction between Flag-hnRNP C1 and PANRNA.Precipitation of PAN RNA is very efficient from UV-irradiated cells (24%;Fig. 15.2, lane 8, top). In contrast, precipitation efficiency is reducedsignificantly in both untreated cells (lane 7) and from cells not expressingFlag-hnRNP C1 (lane 9). Importantly, IP of Flag-hnRNP C1 was similarin both the UV-irradiated and the ‘‘no UV’’ samples (bottom, lanes 7 and8), so the difference in PAN RNA co-IP is not because of differences inantibody accessibility to antigen after irradiation. The intact PAN RNAsignals observed in the supernatant lanes (lanes 4–6) demonstrate that RNAwas not degraded during the IP step; therefore, the lack of signal in thepellet fractions is not due to RNA degradation. The same blot was reprobedfor 7SK RNA (middle), an abundant cellular nuclear RNA (Blencowe,2002). The lack of 7SK RNA co-IP serves as an additional control forspecificity of the hnRNP C–PAN RNA interaction. Taken together, theseresults strongly support the conclusion that hnRNP C1 interacts directlyand specifically with PAN RNA in transiently transfected cells.


3. Cell Mixing Experiment

A second technique used to verify the existence of an RNA–proteininteraction in vivo is the ‘‘cell mixing’’ experiment described by Mili andSteitz (2004). The experimental scheme for this assay is depicted inFig. 15.3A. Six independent transfections (plates A–F) are performed.In plate A, both tagged protein (Flag-hnRNP C1) and ligand RNA(PAN RNA) expression constructs are transfected, whereas in plate B, thecorresponding empty vector controls are transfected. The cells from plate Aand plate B are mixed and lysed and the tagged protein is immunoprecipi-tated. Co-IP of the RNA will be observed if the complex forms in vivo andpersists in extract or if the protein binds the RNA after the cells are lysed.To distinguish between these alternatives, a second IP is performed. Oneplate of cells (plate C) is transfected with the ligand RNA expressionconstruct and the tagged protein vector, whereas another (plate D) istransfected with the ligand RNA vector and tagged protein expressionconstruct. After mixing, lysis, and IP, one expects no co-IP of the RNAif the interaction is formed exclusively in vivo. However, if the RNA andprotein factors associate upon lysis, the RNA will co-IP from these extracts.As with any IP experiment, there is also the possibility of nonspecificbinding to the antibody-bead resin. Thus, a final set of transfections isperformed in which neither set of cells expresses the tagged construct(plates E and F). If the result from mixed plates C and D is negative, platesE and F can be omitted. In summary, one can conclude that an interaction isoccurring in vivo if the co-IP efficiency from the cotransfected cell lysates(plates A and B) is significantly greater than that observed for the mixedsamples (plates C and D).

One advantage of this protocol is that it can be done using relativelygentle cell lysis procedures, which decreases the likelihood of RNA degra-dation and may allow for other downstream analyses. In Section 5, thepossibility of adapting this protocol to uncover protein-binding sites on theRNA is considered. This procedure is the simplest of the three techniquesexamined here since there are no cross-linking conditions to optimize nor isthere a requirement that RNAs be stripped from proteins in extract, as is thecase for the cross-linking based protocols. Finally, the gentle lysis procedureallows the use of antibodies that cannot withstand the harsh treatments usedin cross-linking procedures. Unfortunately, this advantage is limited by amajor disadvantage of the protocol—the cell mixing technique can rarely beapplied to endogenous RNA–protein interactions. Some procedural varia-tions using inducible genes (Mili and Steitz, 2004) or interspecies cell mixes( Jonson et al., 2007; Kittur et al., 2006) are possible, but in the majority ofcases, exogenous expression is necessary for this assay. In contrast to UVcross-linking methods, one cannot determine whether an interaction

A

A+

B

A+

B

A+

B

C+

D

C+

D

C+

D

E+

F

E+

F

E+

F

Input Supe Pellet

PAN

Control

7SK

B

% PAN IP 2.8 0.20.3

Flag-C1

1 2 3 4 5 6 7 8 9

Plate A Plate B Plate C Plate D Plate E Plate FLigand RNA:

Tagged protein:Ligand vector:Protein vector:

Mix: A+B Mix: C+D Mix: E+F

Lyse cells, IP

++−−

−−++

+−−+

−++−

−−++

+−−+

Lyse cells, IP Lyse cells, IP

Figure 15.3 Cell mixing experiment. (A) Experimental scheme; details are describedin the text. (B) Northern andWestern blots show results of a cell mixing experiment.In this case, PAN RNA is the ligand RNA and Flag-hnRNP C1 is the tagged protein.Lane designations refer to the mixed plates depicted in (A).The top and middle panelsareNorthern blots and the bottom is aWestern blot, see Fig.15.2 for details.


observed in this assay is direct or indirect. This may be advantageous if theprotein of interest is part of a larger complex, but does not itself bind directlyto the transcript or disadvantageous if the goal is to determine if the protein–RNA interaction is direct. A final consideration is that the cell mixingexperiment is designed to test whether particular experimental conditionspromote binding of an RNA to a specific protein in cell lysate. If so, theresults do not necessarily exclude the possibility that the interaction alsooccurs in vivo. In this case, conditions can be altered to disrupt binding inextract or in vivo cross-linking procedures can be employed to maintainin vivo interactions.


1. Transfection materials. Use any established protocol.2. Rubber policeman


3. PBS chilled on ice4. Combine cells from plate A with plate B, plate C with plate D, and plate

E with plate F in 15-ml conical tubes.5. RSB100-T-plus [RSB100-T supplemented with 1 mM PMSF, 0.4 U/ml

RNase inhibitor (Promega), competitor RNA (see UV cross-linkingprocedure for comments regarding competitor RNA composition andpreparation)]

6. 15-ml conical centrifuge tubes7. Benchtop centrifuge [Sorvall Legend RTþ (Thermo-Fisher)]8. Microfuge tubes9. Sonicator (Branson Sonifier 450 with a 4.8-mm-diameter tapered

microtip)10. Anti-Flag M2 affinity gel (Sigma), washed three times in RSB100-T.

Use 40 ml of slurry per sample, which corresponds to �20 ml beadvolume. Other tags and their corresponding affinity beads can be sub-stituted, as appropriate.

11. Control RNA (see UV cross-linking procedure)12. Proteinase K solution [0.1 mg/ml proteinase K, 0.1% SDS, 20 mM

Tris–HCl (pH 7.5), 5 mM EDTA, 16.7 ng/ml GlycoBlue (Ambion),0.1 mg/ml torula yeast RNA, 6.7 pg/ml control RNA]. Note: This isthe same mix used in the UV cross-linking experiment, except theconcentrations of proteinase K and SDS are decreased to 0.1 mg/ml and0.1%, respectively.

13. 3 M sodium acetate (pH 5.2)14. PCA

3.2. Procedure

1. Transfect six 60-mm plates (plates A–F) with the same amount of totalDNA using the scheme depicted in Fig. 15.3A. When studying RNAdecay, it is common for a cotransfected protein to regulate the levelsof a putative RNA target. In this case, the amount of the transfectedligand RNA expression construct should be modified to produce simi-lar levels of RNA in the cotransfected (plate A þ B) and mixed cell(plates CþD) lysates. For the experiment shown in Fig. 15.3B, 60-mmplates of HEK293 cells using TransIT-293 (Mirus) were transfectedaccording to the manufacturer’s instructions. Two micrograms of eachplasmid, PAN-WT, pRTS-ORF50, and Flag-hnRNP C1 (or theirvector controls) was transfected for a total of 6 mg per 60-mm plate.

2. Approximately 24 h after transfection, wash cells in 4 ml ice-cold PBS.Be careful to avoid detaching cells from plate.

3. Add 4 ml of ice-cold PBS and harvest cells with a rubber policeman.4. Combine cells from plate A with plates B–D, and plate E with plate F in

15-ml conical tubes.


5. Centrifuge at 700g at 4 �C for 3 min.6. Remove PBS.7. Resuspend cell pellets in 400 ml of RSB100-T-plus and transfer to

microfuge tubes.8. Incubate on ice for 5 min.9. Sonicate the extract. Use the lowest setting for three 5-s bursts; keep

extract on ice for at least 1 min between bursts.10. Centrifuge at 4000g at 4 �C for 10 min.11. Transfer supernatant to a new tube and centrifuge at 10,000g at 4 �C for

10 min.12. Remove 20- and 2.0-ml aliquots and place on dry ice. These are the 5%

RNA and 10% protein ‘‘input’’ samples for Northern and Westernblotting, respectively.

13. Add remaining lysate to washed beads.14. Nutate at 4 �C for 1h.15. Centrifuge at 845g for 1 min at room temperature.16. Remove 20- and 2-ml aliquots of the supernatant and place on dry ice.

These are the 5% RNA and 10% protein ‘‘supernatant’’ samples forNorthern and Western blotting, respectively.

17. Discard remaining supernatant.18. Add 500 ml of ice-cold RSB100-T to beads, centrifuge at 845g for

1 min at room temperature, and discard supernatant. Repeat four timesfor a total of five washes.

19. Resuspend beads in a total volume of 200 ml. Remove 10 ml of slurryand place on dry ice. This is the protein ‘‘pellet’’ sample for Westernblotting.

20. Centrifuge the remaining slurry at 845g for 1 min at room temperatureand discard supernatant.

21. Thaw RNA ‘‘input’’ and ‘‘supernatant’’ samples at room temperature.Add 300 ml proteinase K solution to input, supernatant, and beads.

22. Incubate at 37 �C for 30 min.23. Add 30 ml 3 M sodium acetate (pH 5.2), 350 ml PCA, vortex for 5 to

10 s, and centrifuge at 16,000g for 5 min at room temperature.24. Transfer the upper aqueous layer to a tube containing 900 ml 100%

ethanol; the ethanol mix can be stored indefinitely at –20 �C.25. Analyze RNA using Northern blotting (Fig. 15.3), RNase protection

assay, or RT-PCR. Use the control RNA added to the proteinase Ksolution to normalize samples for loading and recovery (see legend toFig. 15.2 for a description of quantitation).

26. Analyze protein samples using standard Western blot protocols to verifyIP of the protein of interest. Prior to Western analysis, the proteins inthe pellet fractions must be stripped from the beads by boiling for 3 minin standard SDS-PAGE loading buffer.


3.3. Results

Data from a cell mixing experiment are shown in Fig. 15.3B. PAN RNA iscoimmunoprecipitated with Flag-hnRNP C1 only when both areexpressed in the same cells (top, compare lane 7 with lane 8). Moreover,the signal is not a consequence of nonspecific binding of PAN RNA tobeads, as it depends on the presence of Flag-hnRNP C1 (lane 9), and it isspecific for PAN RNA, as 7SK RNA does not coprecipitate with anti-Flagantibodies (middle, lanes 7–9). Importantly, efficient IP of Flag-hnRNP C1is observed from both lysates in which it is present (bottom, lanes 7 and 8).In summary, co-IP of PAN RNA with Flag-hnRNP C1 depends on theircoexpression in the same cells, further supporting the conclusion that PANRNA and hnRNP C1 interact in vivo.

4. RNA Immunoprecipitation

Perhaps themost common technique currently used to examineRNA–protein interactions in vivo is the RNA immunoprecipitation techniquedeveloped by Garcia-Blanco and colleagues (Niranjanakumari et al., 2002).The procedure, based on chromatin immunoprecipitation (ChIP) protocols( Jackson, 1978; Orlando and Paro, 1993; Orlando et al., 1997; Solomon andVarshavsky, 1985), involves chemical cross-linking with formaldehyde totrap complexes formed in vivo. The particular advantage of this protocol isthat, unlike the UV cross-linking procedure described earlier, the cross-linksare reversible. Therefore, increased sensitivity can be achieved by detectingbound RNAs using RT-PCR. An advantage of RIP shared with UV cross-linking is that endogenous RNA–protein complexes can be examinedprovided an antibody is available that is specific to the protein of interestand can withstand the relatively stringent IP conditions used in the protocol.

Covalent cross-links are formed in a two-step process beginning withthe reaction between formaldehyde and an amino group; the resultingSchiff base reacts with a second amino group to form the cross-link(Barker et al., 2005). The general nature of the reaction makes this tech-nique significantly less dependent on the particular conformation of theRNA–protein interface than UV cross-linking. Because both nucleic acid–protein and protein–protein cross-links are produced efficiently, co-IP of aspecific RNA is often not due to a direct interaction. Indeed, the assemblyof RNA-binding proteins has been successfully studied not only by RIP butalso by ChIP analyses (e.g., Arigo et al., 2006; Lei et al., 2001; Listermanet al., 2006; Swinburne et al., 2006). It is clear that the association of RNA-binding proteins with DNA observed in ChIP experiments is sometimesdependent on an RNA bridge (Abruzzi et al., 2004). Thus, the interaction is


not a consequence of the association of the protein directly with DNA oreven with DNA-bound proteins (Abruzzi et al., 2004). Therefore, wheninterpreting RIP data, one must consider that the observed interactions maybe the result of multiple layers of cross-linked factors.


1. Transfection materials, if necessary2. PBS (both room temperature and on ice)3. Methanol-free formaldehyde (Electron Microscopy Sciences). Formal-

dehyde containing methanol is also often used.4. Rocker5. 2 M glycine (pH 7.0)6. Rubber policeman7. 15-ml conical tubes8. Benchtop centrifuge [Sorvall Legend RTþ (Thermo-Fisher)]9. Microfuge tubes10. Microfuges at room temperature and 4 �C11. QIAshredder spin columns (Qiagen) or sonicator (Branson Sonifier 450

with a 4.8-mm-diameter tapered microtip)12. RIPA buffer [1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150

mM sodium chloride, 50 mM Tris–HCl (pH 8.0), 2 mM EDTA]13. RIPA-plus [RIPA supplemented with 10 mM VRC, 1 mM PMSF, and

competitor RNA (see note in UV cross-linking procedure regardingcompetitor RNA)]. Add PMSF immediately before use.

14. RIPA-U (RIPA buffer supplemented with 1 M urea)15. RIPA-U-plus (RIPA-U supplemented with competitor RNA)16. Anti-Flag M2 affinity gel (Sigma), washed three times in RIPA buffer.

Use �40 ml of slurry per sample, which corresponds to �20 ml beadvolume. If using an antibody directed against an endogenous protein, itshould be bound to beads (protein A- or protein G-Sepharose, asnecessary for the particular antibody) using standard protocols(Harlow and Lane, 1988) and washed three times in RIPA buffer.

17. Nutator18. Reverse buffer [10 mM Tris–HCl (pH 6.8), 5 mM EDTA, 10 mM

DTT, 1.0% SDS]19. 2� ProK solution [0.2 mg/ml proteinase K, 40 mMTris–HCl (pH 7.5),

5 mM EDTA, 33.4 ng/ml GlycoBlue (Ambion), 0.2 mg/ml torula yeastRNA, 13.4 pg/ml control RNA]

20. Water baths or dry baths at 37 and 70 �C21. 100 and 70% ethanol22. 2� DNase solution [80 mM Tris–HCl (pH 8.0), 20 mM magnesium

sulfate, 2 mM calcium chloride, 0.2 U/ml RQ1 RNase-free DNase(Promega), 2 U/ml RNasin (Promega)]


23. G-50 buffer [0.25% SDS, 20 mM Tris–HCl (pH 7.0), 0.3 M sodiumacetate, 2 mM EDTA]

24. PCA

4.2. Procedure

1. Transfect cells, if necessary. In the example shown in Fig. 15.4,TransIT-293 was used to transfect each of two 10-cm plates with 4 mgPAN-WT, 4 mg pRTS-ORF50, and 4 mg Flag-hnRNP C1 constructs.One of these plates was cross-linked while the other served as a ‘‘noformaldehyde’’ control. A third plate was transfected with PAN-WT,pRTS-ORF50, and pcDNA3. See UV cross-linking protocol for mod-ifications necessary when using antibodies to endogenous proteins.

2. Approximately 18–24 h posttransfection, wash plates with 10 ml ofroom temperature PBS. Avoid detaching cells from plate. Plates shouldbe �70–100% confluent (�107 cells).

3. Add 10 ml of room temperature PBS to each plate; add methanol-freeformaldehyde to 0.03% to each plate except the ‘‘no formaldehyde’’control. Because the final concentration of formaldehyde can affectresults greatly, it is useful to test a range of concentrations (�0.03–1.0% is reasonable) for each new protein–RNA interaction beinginvestigated. Too much formaldehyde can reduce solubility or maskepitopes, while too little will decrease the likelihood of cross-linking theprotein and RNA of interest. Increased formaldehyde concentrationsmay be necessary for indirect interactions, especially those cases inwhich multiple intermediaries bridge the protein–RNA association.

4. Gently rock for 10 min at room temperature.5. Add 1.25 ml of 2 M glycine (pH 7.0) to quench the reaction.6. Gently rock for 5 min at room temperature.

FormaldehydeFlag-C1 + −+ + −+ + −+

− ++ − ++ − ++Input Supe Pellet

PAN

Control

7SK% PAN IP 0.2 0.01.6

Flag-C1

1 2 3 4 5 6 7 8 9

Figure 15.4 RIPexperiment demonstrating that PANRNAand hnRNPC1are in thesame complex in vivo.The top and middle panels are Northern blots and the bottom is aWestern blot.The details are the same as Fig. 15.2 except that the input and supernatantlanes for bothNorthern andWestern blots are 5%of the total.


7. Harvest cells by scraping with a rubber policeman and transfer to 15-mlconical tubes.

8. Centrifuge at 700g for 3 min at 4 �C.9. Resuspend cells in 10 ml of ice-cold PBS and centrifuge at 700g for

3 min at 4 �C. Discard supernatant and repeat. This step removes anyremaining formaldehyde.

10. Transfer cell pellets to microfuge tubes in 1 ml of ice-cold PBS.11. Centrifuge at 2400g for 1 min at 4 �C.12. Remove PBS (at this point, cell pellets can be frozen on dry ice and

stored at –80 �C).13. Resuspend cell pellet in 400 ml of RIPA-plus.14. Sonicate extract. Use the lowest setting for three 5-s bursts. Keep the

extract on ice for at least 1 min between bursts. Alternatively, addsolution to a QIAshredder spin column. Centrifuge at 16,000g for1 min at 4 �C. Repeat. In some cases, substitution of the QIAshredderstep for sonication leads to increased recovery of intact RNA.

15. Centrifuge at 16,000g for 10 min at 4 �C.16. Transfer supernatant to a new tube and centrifuge at 16,000g for 10 min.17. Remove 20 ml and place on dry ice. This is the 5% ‘‘input’’ sample.18. Add remaining lysate to washed beads.19. Nutate for 2 h at 4 �C.20. Centrifuge at 845g for 1 min at room temperature.21. Remove 20 ml and place on dry ice. This is the 5% ‘‘supernatant’’

sample.22. Discard remaining supernatant.23. Add 500 ml of ice-cold RIPA to beads, centrifuge at 845g for 1 min at

room temperature, and discard supernatant.24. Add 500 ml of RIPA-U-plus to beads.25. Nutate at room temperature for 10 min.26. Centrifuge at 845g for 1 min at room temperature.27. Remove supernatant and repeat RIPA-U-plus wash (steps 24–26).

Replacing the 1 M urea wash with a high salt wash [1 M NaCl,10 mM Tris–HCl (pH 7.5), 0.5% Triton X-100 plus competitorRNA] has also proved successful. This may be useful if an antibody–antigen interaction is sensitive to urea, but not to high salt. If performinga high salt wash, be sure to include a low salt wash to remove residualSDS prior to adding high salt solutions; otherwise, SDS may precipitate.Similarly, add a low salt wash prior to the final wash with RIPA buffer.These stringent washing procedures were necessary to decrease back-ground in the no formaldehyde sample for the hnRNP C1-PANRNAinteraction investigated here and for the interactions investigatedby Garcia-Blanco and colleagues (Niranjanakumari et al., 2002). It isprobable that many RNA–protein interactions will not require theseharsh procedures to strip away uncross-linked RNA.


28. Add 500 ml RIPA-U to beads, centrifuge at 845g for 1 min at roomtemperature, and discard supernatant. Repeat.

29. Add 500 ml of ice-cold RIPA to beads, centrifuge at 845g for 1 min atroom temperature, and discard supernatant.

30. Thaw the 20-ml input and supernatant samples at room temperature.Add 130 ml of reverse buffer to the input, supernatant, and beads.

31. Reverse cross-links by incubating at 70 �C for 45 min.32. Remove 15 ml of each sample for subsequent Western blot analysis and

store at –80 �C.33. Add 150 ml of 2� ProK solution to the remainder of the samples.34. Incubate at 37 �C for 30 min.35. Add 30 ml of sodium acetate (pH 5.2), 350 ml of PCA, vortex for 5–10 s,

and centrifuge at 16,000g for 5 min at room temperature.36. Transfer upper aqueous layer to a tube containing 900 ml of 100%

ethanol; the ethanol mix can be stored indefinitely at –20 �C.37. Incubate ethanol mix on dry ice for 15 min.38. Centrifuge samples at 16,000g for 15 min at room temperature.39. Wash pellets with 70% ethanol and centrifuge samples at 16,000g for

5 min at room temperature.40. Remove all traces of ethanol, air dry, and resuspend pellet in 20 ml

RNase-free water.41. Add 20 ml of 2� DNase solution.42. Incubate at 37 �C for 45 min.43. Add 180 ml of G-50 buffer, 200 ml of PCA, vortex for 5–10 s, and

centrifuge at 16,000g for 5 min at room temperature.44. Transfer upper aqueous layer to fresh tubes and add 500 ml 100%

ethanol.45. Analyze RNA using Northern blotting (Fig. 15.4), RNase protection

assay, or RT-PCR. Use about one-third of the samples for Northern orRNase protection assays and one-fifth of the samples if performingRT-PCR. For Northern blotting, the DNase steps can be omitted.The control RNA added to the proteinase K solution can be used tonormalize samples for loading and recovery for quantitative analyses.See Fig. 15.2 legend for details of quantitation.

46. Protein samples should also be analyzed using standard Western blotprotocols to verify efficient IP of the protein of interest. Prior toWesternanalysis, the proteins in the pellet fractions must be stripped from thebeads by boiling for 3 min in standard SDS-PAGE loading buffer.

4.3. Results

Results of a RIP experiment are shown in Fig. 15.4. PANRNA coimmuno-precipitates with hnRNP C1 more efficiently in the formaldehyde-treatedsample than in the untreated sample (�8-fold; compare lane 8 to lane 7), and


the precipitation is not due to nonspecific binding to beads (lane 9).Importantly, the formaldehyde treatment does not interfere with theantibody–epitope interaction, as the Flag-hnRNP C1 protein precipitateswith similar efficiency in both untreated and cross-linked samples (bottom,lanes 7 and 8). These results support the conclusion that PAN RNA andhnRNP C1 are in the same complex in vivo.

Interestingly, 7SK RNA is also enriched in the cross-linked samples(Fig. 15.4; 0.6% IP compared with 0.1% for both the no formaldehydeand no Flag-hnRNP C1 controls; data not shown). This result can beinterpreted in at least two ways. The simplest interpretation is thathnRNP C1 is in a complex with 7SK RNA in vivo. Indeed, althoughhnRNP C1 was not shown to bind 7SK RNA, other hnRNP proteinshave been reported to interact with 7SK RNA (Hogg and Collins, 2007).Because PAN RNA is expressed in these cells, it is further plausible thatPAN RNA mediates the interaction. If the hnRNP C1-7SK RNA RIPresult reveals a bona fide complex, the lack of detection of the interactionin the UV cross-linking experiment (Fig. 15.2) may indicate that theinteraction is indirect. Moreover, perhaps the putative hnRNP C1–7SKRNA complexes are not detected in the cell mixing experiment becausethey are insoluble in the absence of SDS and are not present in theselysates. Alternatively, the detection of 7SK RNA in the cross-linkedpellets may be due to the fact that both hnRNP C1 and 7SK RNA arevery abundant nuclear factors closely associated with early events in RNAbiogenesis (Blencowe, 2002; Mili et al., 2001). In this model, the detectedinteractions are because of formaldehyde cross-links between multiplemediating factors and are largely a result of their proximity and abun-dance. With current data, it is impossible to distinguish between thesehypotheses.

5. Discussion

Data presented here conclusively demonstrate an interaction betweenKSHV PAN RNA and Flag-hnRNP C1 in transiently transfected culturedcells. The binding is direct, as the interaction is observed in UV cross-linking experiments (Fig. 15.2). Future experiments will test whetherendogenous hnRNP C binds PAN RNA in KSHV-infected cells. Fortu-nately, an hnRNP C-specific antibody has been described that efficientlyimmunoprecipitates hnRNP C in stringent conditions (Choi and Dreyfuss,1984). Therefore, the hnRNP C–PAN RNA interaction can be examinedin KSHV-infected cells by adapting the UV cross-linking and RIP protocolsdescribed here.


The biological consequences of the hnRNP C–PAN RNA interactionremain ambiguous, but there are at least two models that implicatehnRNP C binding in the control of PAN RNA stability. The ENE is a79-nucleotide RNA element in PAN RNA that is necessary and sufficientto increase the nuclear abundance of intronless transcripts (Conrad andSteitz, 2005). The ENE inhibits deadenylation in vitro, inhibits RNA decayin a polyadenylate [poly(A)] tail-dependent fashion in vivo, and interactswith the poly(A) tail in cis (Conrad et al., 2006). Moreover, mutations thatabrogate the poly(A) tail–ENE interaction do not support nuclear accumu-lation of PAN RNA (Conrad et al., 2007). These data support the proposedmodel that the ENE interaction with the poly(A) tail protects the transcript30 end from exonucleases. Because hnRNP C has been shown to promoteRNA annealing (Portman and Dreyfuss, 1994), perhaps it promotes hybri-dization between the U-rich loop of the ENE and the poly(A) tail.Indeed, while the ENE can inhibit deadenylation in the absence of protein,its activity is enhanced in the nuclear extract (Conrad et al., 2006).Alternatively, hnRNP C may retain PAN RNA in the nucleus whereit is sequestered from rapid cytoplasmic RNA decay. This model isconsistent with the observation that hnRNP C is a nonshuttling RNA-binding protein with a dominant nuclear retention signal (Nakielny andDreyfuss, 1996).

The hypothesis that hnRNP C binds PAN RNA in vivo was driven bythe observation that the ENE cross-links to hnRNP C in the nuclear extract(N. K. Conrad and J. A. Steitz, unpublished observation). However, theexperiments described here do not address the site(s) on PAN RNA forhnRNP C binding. Modified versions of the protocols described here arebeing developed to examine this issue. For example, the lysis conditionsused in the cell mixing experiment have already been shown to supportRNase H activity (Conrad et al., 2006, 2007). An oligonucleotide-directedRNase H cleavage step can be performed prior to IP, and only the fragmentof RNA bound by Flag-hnRNP C1 will be coimmunoprecipitated.By repeating this protocol using different oligonucleotides, one can delin-eate the approximate binding site of the protein. Preliminary data suggestthat this strategy will be useful, but that the increased time in extractnecessary for RNase H cleavage leads to further reassociation. Therefore,it is critical to maintain the ‘‘mixed cell’’ controls (Fig. 15.3A; plate CþD)throughout the procedure. The stringent conditions used in the UV cross-linking and RIP procedures exclude this strategy, but it may be possible toperform an RNase H cleavage step after the complexes are bound to thecolumn. In this case, the precipitated complexes would be washed withRNase H buffer and treated with RNase H in the presence of an appropri-ate oligonucleotide. After cleavage and further washing, only the cross-linked RNA fragment will remain bound to the beads. Finally, one canimagine a RIP variation that integrates the extensive nucleic acid shearing


performed in conventional ChIP assays (Orlando et al., 1997). Once theaverage transcript size is significantly shorter than the length of the RNA ofinterest, one can RT-PCR amplify different regions of the transcript anddetermine which gives the highest signal in the bound fraction. Thisfragment presumably contains the binding site for the protein of interest.These modifications will expand the usefulness of the protocols describedhere in the analysis of in vivo RNA–protein interactions.

Of the three protocols described here, UV cross-linking yields thehighest co-IP efficiency of PAN RNA with hnRNP C. However, this ispresumably due to the fact that hnRNP C binds to stretches of uridines(Gorlach et al., 1994), the most efficiently UV-cross-linked base (Williamsand Konigsberg, 1991) and is unlikely to be a general observation.The results in all three assays are also aided greatly by the knowledge andavailability of a high-affinity competitor RNA, poly-U. Anecdotal observa-tions suggest that decreasing the signal in the negative controls is often themost difficult facet of these experiments. Inclusion of poly-U in the cellextracts decreased the background signal in all three assays significantly (datanot shown). These results were also aided greatly by the abundance of bothhnRNP C and PANRNA. Other complexes may not be abundant enoughto examine by Northern blotting and may require more sensitive techniquessuch as quantitative RT-PCR. Unfortunately, as mentioned earlier, thismay preclude the use of UV cross-linking. The applicability of each of theseprotocols to examine an RNA–protein interaction in vivo will depend onthe nature of the particular complex and goals of the researcher.

6. Concluding Remarks

Identification of the protein factors that bind to and regulate mRNAstability in vivo is essential to our understanding of the control of geneexpression. While not limited to RNA decay studies, each of the threeexperiments described here can convincingly demonstrate that a specificprotein interacts with a specific RNA in vivo. UV cross-linking resultsfurther provide proof that an RNA–protein interaction is direct, but theprocedure may not be applicable to every RNA–protein interaction.Cell mixing experiments are the easiest to perform, but have the majordisadvantage of not being applicable to endogenous RNA–protein interac-tions. RIP has the advantage of the use of RT-PCR for detection, a verysensitive technique. In addition, variations of the RIP and UV cross-linkingprotocols have recently been developed that screen globally for RNApartners of a specific protein (Keene et al., 2006; Ule et al., 2005).The procedures described here, UV cross-linking, cell mixing, and RIP,are powerful tools used for assaying RNA–protein interactions in vivo.


ACKNOWLEDGMENTS

These experiments were initiated in the laboratory of Dr. Joan Steitz. I thank Dr. Steitz forher support, guidance, and encouragement. I thank Drs. Stavroula Mili and Heidi Cook forthe cell mixing and UV cross-linking protocols, respectively, upon which the proceduresdescribed here are based. I thank Brooke Belyea and Crystal Dobson for technical assistancewith the experiments presented here.

REFERENCES

Abruzzi, K. C., Lacadie, S., and Rosbash, M. (2004). Biochemical analysis of TREXcomplex recruitment to intronless and intron-containing yeast genes. EMBO J. 23,2620–2631.

Arigo, J. T., Carroll, K. L., Ames, J. M., and Corden, J. L. (2006). Regulation of yeastNRD1 expression by premature transcription termination. Mol. Cell 21, 641–651.

Barker, S., Weinfeld, M., and Murray, D. (2005). DNA-protein crosslinks: Their induction,repair, and biological consequences. Mutat. Res. 589, 111–135.

Barreau, C., Paillard, L., and Osborne, H. B. (2005). AU-rich elements and associatedfactors: Are there unifying principles Nucleic Acids Res. 33, 7138–7150.

Bergman, N., Moraes, K. C., Anderson, J. R., Zaric, B., Kambach, C., Schneider, R. J.,Wilusz, C. J., and Wilusz, J. (2007). Lsm proteins bind and stabilize RNAs containing50 poly(A) tracts. Nat. Struct. Mol. Biol. 14, 824–831.

Blencowe, B. J. (2002). Transcription: Surprising role for an elusive small nuclear RNA.Curr. Biol. 12, R147–R149.

Brennan, C. M., and Steitz, J. A. (2001). HuR and mRNA stability. Cell. Mol. Life Sci. 58,266–277.

Chang, P. J., Shedd, D., Gradoville, L., Cho, M. S., Chen, L. W., Chang, J., and Miller, G.(2002). Open reading frame 50 protein of Kaposi’s sarcoma-associated herpesvirusdirectly activates the viral PAN and K12 genes by binding to related response elements.J. Virol. 76, 3168–3178.

Choi, Y. D., and Dreyfuss, G. (1984). Monoclonal antibody characterization of the Cproteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells.J. Cell Biol. 99, 1997–1204.

Coller, J., and Wickens, M. (2002). Tethered function assays using 30 untranslated regions.Methods 26, 142–150.

Conrad, N. K., Mili, S., Marshall, E. L., Shu, M. D., and Steitz, J. A. (2006). Identification ofa rapid mammalian deadenylation-dependent decay pathway and its inhibition by a viralRNA element. Mol. Cell 24, 943–953.

Conrad, N. K., Shu, M. D., Uyhazi, K. E., and Steitz, J. A. (2007). Mutational analysis ofa viral RNA element that counteracts rapid RNA decay by interaction with thepolyadenylate tail. Proc. Natl. Acad. Sci. USA 104, 10412–10417.

Conrad, N. K., and Steitz, J. A. (2005). A Kaposi’s sarcoma virus RNA element thatincreases the nuclear abundance of intronless transcripts. EMBO J. 24, 1831–1841.

Cook, H. L., Mischo, H. E., and Steitz, J. A. (2004). The Herpesvirus saimiri small nuclearRNAs recruit AU-rich element-binding proteins but do not alter host AU-rich element-containing mRNA levels in virally transformed T cells. Mol. Cell. Biol. 24, 4522–4533.

Dean, J. L., Sully, G., Clark, A. R., and Saklatvala, J. (2004). The involvement of AU-richelement-binding proteins in p38 mitogen-activated protein kinase pathway-mediatedmRNA stabilisation. Cell Signal 16, 1113–1121.


Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993). hnRNP proteins andthe biogenesis of mRNA. Annu. Rev. Biochem. 62, 289–321.

Fan, X. C., Myer, V. E., and Steitz, J. A. (1997). AU-rich elements target small nuclearRNAs as well as mRNAs for rapid degradation. Genes Dev. 11, 2557–2568.

Garneau, N. L., Sokoloski, K. J., Opyrchal, M., Neff, C. P., Wilusz, C. J., and Wilusz, J.(2007). The 30 untranslated region of Sindbis virus represses the deadenylation of viraltranscripts in mosquito and mammalian cells. J. Virol. 82, 880–892.

Gorlach, M., Burd, C. G., and Dreyfuss, G. (1994). The determinants of RNA-bindingspecificity of the heterogeneous nuclear ribonucleoprotein C proteins. J. Biol. Chem.269, 23074–23078.

Harlow, E., and Lane, D. (1988). ‘‘Antibodies: A Laboratory Manual’’ Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY.

Hogg, J. R., and Collins, K. (2007). RNA-based affinity purification reveals 7SKRNPs withdistinct composition and regulation. RNA 13, 868–880.

Hosoda, N., Lejeune, F., and Maquat, L. E. (2006). Evidence that poly(A) binding proteinC1 binds nuclear pre-mRNA poly(A) tails. Mol. Cell. Biol. 26, 3085–3097.

Jackson, V. (1978). Studies on histone organization in the nucleosome using formaldehyde asa reversible cross-linking agent. Cell 15, 945–954.

Jonson, L., Vikesaa, J., Krogh, A., Nielsen, L. K., Hansen, T., Borup, R., Johnsen, A. H.,Christiansen, J., and Nielsen, F. C. (2007). Molecular composition of IMP1 ribonucleo-protein granules. Mol. Cell. Proteomics 6, 798–811.

Karijolich, J., Stephenson, D., and Yu, Y. T. (2007). Biochemical purification of boxH/ACA RNPs involved in pseudouridylation. Methods Enzymol. 425, 241–262.

Keene, J. D. (1996). RNA recognition by autoantigens and autoantibodies. Mol. Biol. Rep.23, 173–181.

Keene, J. D., Komisarow, J. M., and Friedersdorf, M. B. (2006). RIP-Chip: The isolationand identification of mRNAs, microRNAs and protein components of ribonucleopro-tein complexes from cell extracts. Nat. Protoc. 1, 302–307.

Kittur, N., Darzacq, X., Roy, S., Singer, R. H., and Meier, U. T. (2006). Dynamicassociation and localization of human H/ACA RNP proteins. RNA 12, 2057–2062.

Krecic, A. M., and Swanson, M. S. (1999). hnRNP complexes: Composition, structure, andfunction. Curr. Opin. Cell Biol. 11, 363–371.

Lei, E. P., Krebber, H., and Silver, P. A. (2001). Messenger RNAs are recruited for nuclearexport during transcription. Genes Dev. 15, 1771–1782.

Lerner, M. R., Boyle, J. A., Hardin, J. A., and Steitz, J. A. (1981). Two novel classes of smallribonucleoproteins detected by antibodies associated with lupus erythematosus. Science211, 400–402.

Lerner, M. R., and Steitz, J. A. (1979). Antibodies to small nuclear RNAs complexed withproteins are produced by patients with systemic lupus erythematosus. Proc. Natl. Acad.Sci. USA 76, 5495–5499.

Listerman, I., Sapra, A. K., and Neugebauer, K. M. (2006). Cotranscriptional coupling ofsplicing factor recruitment and precursor messenger RNA splicing in mammalian cells.Nat. Struct. Mol. Biol. 13, 815–822.

Lykke-Andersen, J., Shu, M. D., and Steitz, J. A. (2000). Human Upf proteins target anmRNA for nonsense-mediated decay when bound downstream of a termination codon.Cell 103, 1121–1131.

Mayrand, S., and Pederson, T. (1981). Nuclear ribonucleoprotein particles probed in livingcells. Proc. Natl. Acad. Sci. USA 78, 2208–2212.

Meisenheimer, K. M., and Koch, T. H. (1997). Photocross-linking of nucleic acids toassociated proteins. Crit. Rev. Biochem. Mol. Biol. 32, 101–140.


Mili, S., Shu, H. J., Zhao, Y., and Pinol-Roma, S. (2001). Distinct RNP complexes ofshuttling hnRNP proteins with pre-mRNA and mRNA: Candidate intermediates information and export of mRNA. Mol. Cell. Biol. 21, 7307–7319.

Mili, S., and Steitz, J. A. (2004). Evidence for reassociation of RNA-binding proteins aftercell lysis: Implications for the interpretation of immunoprecipitation analyses. RNA 10,1692–1694.

Myer, V. E., Fan, X. C., and Steitz, J. A. (1997). Identification of HuR as a proteinimplicated in AUUUA-mediated mRNA decay. EMBO J. 16, 2130–2139.

Nakielny, S., and Dreyfuss, G. (1996). The hnRNP C proteins contain a nuclear retentionsequence that can override nuclear export signals. J. Cell Biol. 134, 1365–1373.

Niranjanakumari, S., Lasda, E., Brazas, R., and Garcia-Blanco, M. A. (2002). Reversiblecross-linking combined with immunoprecipitation to study RNA-protein interactionsin vivo. Methods 26, 182–190.

Orlando, V., and Paro, R. (1993). Mapping Polycomb-repressed domains in the bithoraxcomplex using in vivo formaldehyde cross-linked chromatin. Cell 75, 1187–1198.

Orlando, V., Strutt, H., and Paro, R. (1997). Analysis of chromatin structure by in vivoformaldehyde cross-linking. Methods 11, 205–214.

Penalva, L. O., and Keene, J. D. (2004). Biotinylated tags for recovery and characterizationof ribonucleoprotein complexes. Biotechniques 37, 604, 606, 608–610.

Peng, S. S., Chen, C. Y., Xu, N., and Shyu, A. B. (1998). RNA stabilization by the AU-richelement binding protein, HuR, an ELAV protein. EMBO J. 17, 3461–3470.

Portman, D. S., and Dreyfuss, G. (1994). RNA annealing activities in HeLa nuclei. EMBO J.13, 213–221.

Reed, R., and Chiara, M. D. (1999). Identification of RNA-protein contacts withinfunctional ribonucleoprotein complexes by RNA site-specific labeling and UV cross-linking. Methods 18, 3–12.

Solomon, M. J., and Varshavsky, A. (1985). Formaldehyde-mediated DNA-protein cross-linking: A probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82,6470–6474.

Song, M. J., Li, X., Brown, H. J., and Sun, R. (2002). Characterization of interactionsbetween RTA and the promoter of polyadenylated nuclear RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8. J. Virol. 76, 5000–5013.

Srisawat, C., and Engelke, D. R. (2002). RNA affinity tags for purification of RNAs andribonucleoprotein complexes. Methods 26, 156–161.

Sun, R., Lin, S. F., Gradoville, L., and Miller, G. (1996). Polyadenylated nuclear RNAencoded by Kaposi sarcoma-associated herpesvirus. Proc. Natl. Acad. Sci. USA 93,11883–11888.

Swinburne, I. A., Meyer, C. A., Liu, X. S., Silver, P. A., and Brodsky, A. S. (2006).Genomic localization of RNA binding proteins reveals links between pre-mRNAprocessing and transcription. Genome Res. 16, 912–921.

Tenenbaum, S. A., Lager, P. J., Carson, C. C., and Keene, J. D. (2002). Ribonomics:Identifying mRNA subsets in mRNP complexes using antibodies to RNA-bindingproteins and genomic arrays. Methods 26, 191–198.

Ulbrich, N., Wool, I. G., Ackerman, E., and Sigler, P. B. (1980). The identification byaffinity chromatography of the rat liver ribosomal proteins that bind to elongator andinitiator transfer ribonucleic acids. J. Biol. Chem. 255, 7010–7019.

Ule, J., Jensen, K., Mele, A., and Darnell, R. B. (2005). CLIP: a method for identifyingprotein-RNA interaction sites in living cells. Methods 37, 376–386.

Vasudevan, S., and Steitz, J. A. (2007). AU-rich-element-mediated upregulation of transla-tion by FXR1 and Argonaute 2. Cell 128, 1105–1118.


Wagenmakers, A. J., Reinders, R. J., and van Venrooij, W. J. (1980). Cross-linking ofmRNA to proteins by irradiation of intact cells with ultraviolet light. Eur. J. Biochem.112, 323–330.

Williams, K. R., and Konigsberg, W. H. (1991). Identification of amino acid residues atinterface of protein-nucleic acid complexes by photochemical cross-linking. MethodsEnzymol. 208, 516–539.

Wilson, G. M., and Brewer, G. (1999). Identification and characterization of proteinsbinding A þ U-rich elements. Methods 17, 74–83.

Yamashita, A., Chang, T. C., Yamashita, Y., Zhu, W., Zhong, Z., Chen, C. Y., andShyu, A. B. (2005). Concerted action of poly(A) nucleases and decapping enzyme inmammalian mRNA turnover. Nat. Struct. Mol. Biol. 12, 1054–1063.

Zhong, W., Wang, H., Herndier, B., and Ganem, D. (1996). Restricted expression ofKaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.Proc. Natl. Acad. Sci. USA 93, 6641–6646.

Documents

[Methods in Enzymology] RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways Volume 449 || Co-Immunoprecipitation Techniques for Assessing RNA–Protein