Plant Molecular Biology 46: 79–88, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Identification of a cytoskeleton-associated 120 kDa RNA-binding proteinin developing rice seeds

R. Sami-Subbu1, Sang-Bong Choi, Yujia Wu, Changlin Wang and Thomas W. Okita∗Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 USA (∗author forcorrespondence; e-mail: t.okita@wsu.edu); 1present address: National Chemical Laboratory, Pune 411 008, India

Received 15 May 2000; accepted in revised form 13 November 2000

Key words: cytoskeleton, protein bodies, RNA-binding protein, rice prolamine, Rp120

Abstract

During rice seed development, prolamine RNAs are localized to the surface of the prolamine storage protein bodies(PBs), organelles bounded by the endoplasmic reticulum (ER). The exact mechanism by which prolamine RNAsare enriched on this ER subdomain is not known but recent evidence indicates the directed transport and targetingof prolamine RNAs to the prolamine PBs. As such a process involves RNA signal determinants and cytoskeleton-interacting proteins that recognize these signals, we obtained an enriched cytoskeleton-PB fraction and identifieda prominent RNA-binding activity, Rp120, by RNA-binding UV-cross-linking assay. Recombinant cDNA clonesof Rp120 revealed that the primary sequence shared considerable structural homology to the human transcriptionalcoactivator p100 and possessed a modular organization, four nucleic acid-binding SN domains, a tudor domainand a coil-coil domain. Consistent with the presence of SN domains, Rp120 binds a variety of RNAs includingprolamine RNA. Interaction with the latter RNA, however, was specific as binding activity was evident only to theprolamine 3′ UTR and not to the 5′ UTR or coding sequences. Rp120 is also able to interact with other proteins asits sedimentation behavior in sucrose density gradient suggests an association with the cytoskeleton. The presenceof a tudor domain, suggested to have a role in RNA processing or transport, together with the SN and coiled-coildomains are consistent with the view that Rp120 may be involved in RNA sorting in rice endosperm.

Abbreviations: DAF, days after flowering; ER, endoplasmic reticulum; PB, protein body; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SN, staphylococcal nuclease; UTR, untranslated region

Introduction

Rice endosperm cells synthesize and accumulate twomajor classes of storage proteins; prolamines, the stor-age protein typically accumulated by cereals, as wellas glutelins, proteins related to the 11S globulins typ-ically observed in dicots (Muench and Okita, 1997;Muench et al., 1999; Takaiwa et al., 1999). Althoughthe mRNAs for both storage proteins are translatedon the rough ER membrane, results from biochemicalstudies suggest that these storage protein mRNAs arenot randomly distributed on this membrane complexbut rather distributed to distinct ER subdomains. Pro-lamine transcripts are highly enriched within a purifiedprolamine protein body PB fraction whereas glutelin

transcripts predominate in enriched microsomal mem-brane fractions (Yamagata and Tanaka, 1986; Kim etal., 1993). These initial observations were confirmedby Li et al. (1993) who showed, by high-resolutionin situ hybridization techniques, that prolamine tran-scripts are localized on the PB-ER, while glutelintranscripts predominate on the cisternal ER. More re-cent in situ RNA localization studies indicate thatprolamine RNAs are exclusively localized to the pro-lamine PBs by an RNA-based mechanism (Choi et al.,2000).

The targeting of RNAs to specific intracellular re-gions is now a well-established phenomenon in manytypes of eukaryotic cells including plants. In the betterunderstood animal systems, RNA sorting involves a

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stepwise localization pathway involving transport ofthe mRNA and anchoring to a specific site (Wilhelmand Vale, 1993; St Johnson, 1995; Hesketh, 1996;Singer, 1996). Both of these cellular processes relyon components of the cytoskeleton. For example, mi-crotubules are involved in the transport of Vg1 mRNAto the vegetal half of the oocyte while microfilamentsare important for anchoring of the RNA to the cortex(Yisraeli et al., 1990). Similarly, Ainger et al. (1997)demonstrated that microinjected myelin basic proteinRNAs form granules within minutes and move alongmicrotubule tracks where they are dispersed to theperipheral membranes of oligodendrocytes. Microfil-aments have been suggested to be responsible for bothtransport and anchoring of actin RNA (Singer, 1992).

In rice endosperm cells, the segregation of pro-lamine and glutelin storage protein mRNAs to distinctER subdomains is an attractive model system to studythe transport and localization of mRNAs especiallysince these transcripts are abundantly expressed inseed. Okita and colleagues (Okita et al., 1994; Okitaand Rogers, 1996) suggested three possible mech-anisms by which prolamine RNAs are localized tothe prolamine PBs. These included sorting of thepolysome complex to the PBs by nascent peptide sig-nals, sorting of the RNA itself or stabilization of pro-lamine polysome complex on the PBs by a cross-talkprocess. Recent analysis of the intracellular location ofsynthetic prolamine transgenes expressed during riceseed development indicates that prolamine RNA lo-calization to the prolamine PBs is dependent on RNAsignal determinants (Choi et al., 2000). Hence, pro-lamine transcripts are transported from the nucleus tonear or on the surface of the prolamine PBs.

An RNA-based mechanism infers the involvementof the cytoskeleton. Indeed, evidence has been ob-tained supporting the cytoskeletal association of pro-lamine polysomes to the PBs (Muench et al., 1998).Polysomes are bound to the PB surface by a detergent-resistant (i.e., non-membrane) but salt-sensitive man-ner, properties consistent with the involvement ofthe cytoskeleton (Davies et al., 1991, 1993, 1996;Abe and Davies, 1995; Muench et al., 1998). Di-rect visual evidence for this association has beenobtained. Confocal microscopic-immunofluorescentstudies show that the PBs are closely associated withboth microtubules and microfilaments (Muench et al.,2000).

Recent studies are beginning to decipher the sig-nals and mechanisms responsible for mRNA localiza-tion. The targeting signals of bicoid (Macdonald and

Kerr, 1997) and Vg1 (Deshler et al., 1998) RNA spansseveral hundred nucleotides suggesting that multiplecis elements for protein binding are responsible forthe various steps in RNA movement and anchoringto specific regions of the cell. The localization ofprolamine RNAs to the PB-ER by an RNA-basedmechanism infers the existence of RNA-binding pro-teins that recognize one or more ‘zip code’ signalswithin the prolamine RNA sequence. We, therefore,undertook a study to identify the RNA-binding activ-ities in an enriched cytoskeleton-PB fraction. RNAUV-crosslinking experiments showed that the puri-fied fractions obtained from Poly(U)-Sepharose andsuccessive heparin-agarose column chromatographyrevealed the presence of RNA-binding activities in thecytoskeleton-PB fraction. One of the RNA-bindingactivities was a 40 kDa protein which recognizes an A-rich region of 5′ UTR and 3′ UTR of prolamine RNAas well as the 3′ UTR of glutelin mRNA but not theircoding sequences (Sami-Subbu et al., 1999).

In the present study, we report the isolation andcharacterization of a second RNA-binding activity,Rp120, from the cytoskeleton-PB-enriched fraction.Rp120 binds to several different RNA substrates al-though binding preference is restricted to the 3′ UTRfor the prolamine RNA and not to the 5′ UTR or cod-ing sequence. The expression of Rp120 is restricted tovery young developing seeds and exhibits sedimenta-tion properties consistent with its association with thecytoskeleton. Analysis of the derived coding sequenceof a cDNA clone reveals close structural homologyto the human EBNA-2 coactivator p100. In additionto possessing the four repeat SN and coil-coil do-mains, structural motifs for nucleic acid and proteininteractions, respectively, Rp120 contains a tudor do-main which has been suggested to be involved in RNAtransport or processing (Ponting, 1997).

Materials and methods

Rp120 purification

A cytoskeleton-PB-enriched fraction was obtainedfrom rice (Oryza sativa L. M109) seeds at the milkystage as described elsewhere (Davies et al., 1991)with slight modifications. Developing rice seeds (80 g)were ground in 100 ml of cytoskeleton-stabilizingbuffer (5 mM HEPES pH 7.5, 10 mM magnesium ac-etate, 2 mM EGTA, 1 mM PMSF, 1 mM DTT, 200 mMsucrose and 1% Triton X-100). The extract was passed

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through miracloth and centrifuged at 2000 rpm for 10min with a SS34 rotor in a Sorval centrifuge. Thepellet enriched in cytoskeleton, PB and starch grainswas suspended in 40 ml of cytoskeleton stabilizingbuffer and 7 ml was loaded on sucrose density gra-dients consisting of 14 ml each of 60% and 80%sucrose prepared in 5 mM HEPES pH 7.5 and 10 mMmagnesium acetate. The gradient was spun at 27 000rpm for 90 min with a Beckman SW28 rotor. Thecytoskeleton-PB fraction, which sedimented at the 60–80% interface, was collected and diluted in 25 ml ofhigh-salt buffer (200 mM Tris-HCl pH 8.5, 500 mMpotassium acetate, 2 mM EGTA, 50 mM magnesiumacetate) to disrupt the cytoskeletal network. This high-salt extract was then centrifuged at 49 000 rpm for45 min with a SW50 rotor to remove polysomes andPBs. The supernatant was dialyzed against Poly(U)-Sepharose column loading buffer (20 mM HEPESpH 7.5, 100 mM KCl, 1 mM DTT, 10% glycerol).Cytoskeleton-PB extract containing about 56 mg pro-tein was passed through a Poly(U)-Sepharose column(5 ml total column volume, 1 cm diameter), andwashed with 3 column volumes of loading buffer.Bound proteins were eluted with 300 mM KCl andthen with 1 M KCl. The 300 mM KCl fraction (10 mgprotein) was dialyzed in loading buffer containing100 mM KCl and passed through a heparin-agarosecolumn. The column was washed with 2 to 3 columnvolumes of buffer and bound proteins were eluted with300 mM and 1 M KCl. All column chromatographicfractions and the crude cytoskeleton-PB fraction weresubjected to RNA-protein UV-cross-linking assay.

For antibody production, the 300 mM KCl fraction(130 µg protein) from the heparin-agarose columnwas subjected to SDS-PAGE. The polyacrylamide gelwas slightly stained with Coomassie brilliant bluesolution and the gel section containing Rp120 was col-lected and crushed well. The slurry was mixed withadjuvant (Ribi Immunochem) and injected in rabbitsfor antibody production. A booster injection was given2 weeks after the initial injection.

RNA-protein UV-cross-linking assay

Prolamine 7 (Kim and Okita, 1988), glutelin (Okita etal., 1989) and Xenopus laevis EF1α (Ambion) cDNAplasmids were linearized with either BamHI or withappropriate restriction enzyme to yield a completesense strand RNA in a transcription reaction. Puri-fied template (1 µg) was used in each transcriptionreaction.

Radiolabeled prolamine transcripts were obtainedwith an Ambion Maxiscript transcription kit withslight modifications. The reaction (25 µl) contained0.5 mM each of ATP, CTP and GTP, 40 µm UTP, 100µCi of 32P-UTP (3000 Ci/mmol), 20 units of T3 or T7RNA polymerase and 1 unit of RNase inhibitor (5′-3′Inc.). The reaction was terminated by the addition of 1unit of RNase-free DNase (Promega). A 1 µl reactionwas resolved by urea-PAGE to ensure the synthesis ofcomplete RNA transcript.

The RNA UV-crosslinking reaction contained20 mM HEPES, 100 mM KCl, 1 mM DTT, 10% glyc-erol, 4 µg of yeast tRNA, 1 unit of RNase inhibitor(5′-3′ Inc.), 500 ng of purified protein, and either20 or 100 fmol of radiolabeled RNA. The reactionwas incubated at 4 ◦C for 10 min, transferred ontoa parafilm sheet and then exposed to UV light for 7min using an Ultralum UV crosslinker (Stratagene).SDS-PAGE sample buffer was added and subjected toelectrophoresis. After completion of electrophoresis,the gel was dried and exposed to X-ray film for 12 to48 h.

Subcellular fractionation

Two different sucrose density gradients were used toevaluate the sedimentation behavior of Rp120 underlow- and high-ionic-strength conditions. The low-ionic-strength sucrose density gradient consisted oftwo layers, 4 ml of 60% sucrose and 4 ml of 80%sucrose, prepared in cytoskeleton-stabilizing buffer.A 50–64% sucrose density step gradient was usedto analyze the sedimentation of Rp120 prepared un-der high-ionic conditions (see below) which disruptthe cytoskeleton. The step gradient consisted of 1 mlsucrose layers increasing at 2% intervals. The stepgradient was left in the cold room for 1 to 2 h beforeloading the seed extract.

About 3 g of dehulled rice seeds were gent-ly ground in either cytoskeleton-stabilizing bufferor high-ionic-strength PB extraction buffer (2 ml)consisting of 20 mM HEPES-NaOH pH 7.5,50 mM MgCl2, 100 mM KCl, 200 mM sucrose, 1 mMDTT and 1 mM PMSF. The slurry was passed throughmiracloth and then spun at 37×g twice to remove nu-clei and large starch grains. The resulting high- andlow-ionic-strength supernatants were loaded onto the50–64% and 60–80% sucrose gradients, respectively,and then centrifuged in a SW50 rotor at 32 000 rpmfor 90 min. Different layers from the gradient werecollected, resolved by SDS-PAGE and transferred onto

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nitrocellulose membrane. Membranes were blockedwith 5% milk powder in TBS (10 mM Tris-HCl pH7.2, 150 mM NaCl) and incubated with primary anti-bodies (1:1000) in TBS containing 2% milk powder.After three times of washes in TBS, the blots wereincubated with secondary antibody conjugated withhorseradish peroxidase (HRP). After three times ofwashes with TBS, HRP substrate (Pierce) was spreadon the blots and exposed to X-ray film.

Screening for Rp120 cDNA clones

Immunoscreening of λ phage was performed ac-cording to the manufacturer’s protocol (Stratagene)with slight modifications. About a million plaques ofλZAP II rice mid-developing seed cDNA library werescreened by immunological method. DNA sequencingof two cDNA clones revealed that they were iden-tical and contained about 3.0 kb of DNA sequence.Since northern analysis indicated that Rp120 RNAwas about 3.5–4 kb in size, a second screen of thecDNA library was conducted with a 32P-radiolabeled5′-end DNA fragment of Rp120. Subsequent analy-sis of the positive clones resulted in the isolation of afurther 548 nucleotides, which included the putativeinitiation codon.

Results

Purification of Rp120 from cytoskeleton-enrichedfraction

A cytoskeleton-PB-enriched fraction from developingrice seed was prepared by sucrose density gradientcentrifugation as described previously (Sami-Subbu etal., 1999). After extraction by cytoskeleton-disruptionbuffer and clarification by high-speed centrifugationfollowed by dialysis, the protein extract was subjectedto Poly(U)-Sepharose chromatography. Protein frac-tions were collected at 0.3 and 1.0 M KCl, and thenanalyzed by RNA-protein UV-cross-linking assay. Aprominent 120 kDa RNA-binding activity was ob-served in the 300 mM KCl fraction. Such a bindingactivity was not observed in the crude extract. Minorbinding activities were also evident at 100, 75 and 40kDa. The 1 M KCl fraction showed a strong bindingactivity at 40 kDa which had been characterized inan earlier study (Sami-Subbu et al., 1999). When the300 mM KCl fraction was then subjected to heparin-agarose chromatography, the 120 kDa binding activity

together with a few weak activities at 100 and 75 kDaeluted off at 300 mM KCl.

The extent of Rp120 binding to 32P-radiolabeledprolamine RNA was dependent on the amount ofprobe used in the UV-cross-linking reaction. When 20fmol of labeled RNA was used in the cross-linking re-action, a strong binding activity at 40 kDa was readilydetected whereas a very weak reaction was observedfor the 120 kDa protein. However, when the amountof 32P-radiolabeled RNA probe was increased to 100fmol, a much stronger Rp120 binding activity wasevident whereas the binding activity of the 40 kDapolypeptide remained unchanged. These results in-dicate that the 120 kDa RNA-binding activity has amuch lower affinity for the probe than the 40 kDaRNA-binding activity.

Rp120 possesses a multidomain organization

Analysis by one- or two-dimensional PAGE of the300 mM KCl fraction obtained from the heparin-agarose column showed only a single polypeptideband at 120 kDa. This feature enabled us to obtainsufficient quantities of Rp120 for the production ofantibodies. This antibody cross-reacted to the native120 kDa band (data not shown). cDNA clones forRp120 were then isolated by immunological screeningof a developing rice seed cDNA library. Two positiveclones were obtained and subsequently determined tobe identical based on DNA sequence analysis. Theisolated cDNA was about 3.0 kb in length and lackedan AUG initiation codon. Since northern blot analy-sis of developing seed RNA indicated that the Rp120transcript was about 3.5 to 4.0 kb in length (datanot shown), additional screening of the cDNA li-brary was conducted using a 5′-end probe. Severalclones were identified which contained an additional548 nucleotides in the 5′ end of the Rp120 transcriptsequences. Analysis of the total cDNA sequence re-vealed the presence of a single open reading frame of986 amino acid residues beginning with an AUG startcodon.

A search of the database showed that the Rp120shares about 30% identity (ignoring gaps) with thehuman nuclear transcriptional co-activator p100, aprotein conserved from yeast to mammals. Structuralanalysis of Rp120 showed that, like p100, it is com-posed of series of modular domains (Figure 1A). Oneprominent domain is the repeated SN domain (Fig-ure 1B). Callebaut and Moron (1997) have shownthat each of these repeats is analogous to Staphylo-

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Figure 1. The multidomain organization of Rp120 (A), and primary sequence comparison of SN (B) and tudor (C) domains with other knownsequences. A. A schematic diagram of Rp120 based on SMART (Schultz et al., 1998) is depicted. B. Alignment of the four repeated SNdomains located within Rp120 with homologous sequences from S. hyicus and S. aureus SN nuclease. Subdomains A and B correspond to theoligonucleotide/oligosaccharide-binding (OB)-fold and SN-fold respectively (Murzin, 1993; Callebaut and Mornon, 1997). C. Comparison oftudor domain of Rp120 to tudor domains found in other proteins (adapted from Ponting et al., 1997). The conserved motifs x, y and z of thetudor domain are boxed. p100 (human) (Tong et al., 1995); human p100 homologue, C. elegans p100 (GenBank accession number D72470);SMN, human survival motor neuron (Lefebvre et al., 1995); AKAP (human), human A-kinase anchor protein 149 (Trendelenburg et al., 1996);homeless (Drom), Drosophila homeless gene (Gillespie and Berg, 1995); tudor-Drom1–4, tudor domains 1–4 out of 10 tudor domains withinthe Drosophila posterior group gene tudor (Golumbeski et al., 1991).

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Figure 2. The binding specificity of Rp120 to different RNAs asshown by RNA-protein UV-cross-linking assays. A. Rp120 bindingreactions to 32P-labeled prolamine RNA in the absence or presenceof competitor RNA. All lanes contain 100 fmol of 32P-radiolabeledRNA and protein from the 300 mM KCl fraction from the he-parin-agarose column except for lane 1which is a control reactionwithout added protein. Lanes 2 and 3 contained 50× and 100×,respectively, of excess unlabeled prolamine RNA. Lanes 4 and5 contained 50× and 100×, respectively, of excess unlabeledprolamine RNA lacking the 3′ UTR + poly(A) tail. B. Rp120 bind-ing to various segments of 32P-radiolabeled prolamine RNA (20fmol/reaction). Lane 1, 32P-labeled 5′ UTR; lane 2, 32P-labeledcoding sequence; lane 3, 32P-labeled 3′ UTR + poly(A) tail; lane 4,32P-labeled 3′ UTR; lane 5, BSA control. C. Rp120 binding to vari-ous 32P-labeled RNAs (20 fmol/reaction). Lane 1, 32P-labeled pro-lamine RNA; lane 2, 32P-labeled glutelin RNA; lane 3, 32P-labeledEF1α RNA.

coccus nuclease (SN) fold, which functions to bindsingle-stranded nucleic acids. Other prominent struc-tures are the tudor domain (residues 756–840) and aC-terminal coiled-coil motif (Figure 1A). The tudordomain is a repetitive motif observed in the maternallyencoded TUDOR protein, which is required for pos-terior pole formation during oogenesis in Drosophilaembryo. HCA analysis indicates three motifs, x, y andz, present in the TUDOR and human p100 proteins(Callebaut and Mornon, 1997). The x, y and z motifsare also conserved in Rp120. Consequently, Rp120shares many of the structural features of p100 in con-taining four SN folds, and a tudor domain followedby a C-terminal coiled-coil domain. The capacity forRNA binding (SN domains) and protein-to-protein in-teractions (coiled-coil domain) were further studied asdiscussed below.

Rp120 binds to several different mRNAs but showssequence specificity

The substrate specificity of Rp120 was studied usingdifferent mRNAs as probes. Radiolabeled prolamine,glutelin and Xenopus laevis EF1α RNAs were incu-

bated with the 300 mM KCl heparin-agarose fraction,and the extent of binding assessed by UV-crosslinkingfollowed by gel retardation analysis. The Rp120 notonly binds to prolamine RNA but also to glutelin andEF1α RNAs (Figure 2C). The interaction with severaldifferent RNAs indicated either that Rp120 is a generalRNA-binding protein or that it recognizes a commonnucleotide motif present in these RNA transcripts. Pre-vious studies with the 40 kDa RNA-binding activityalso showed that this protein interacted with multipleRNA species but that binding activity was dependenton the presence of specific sequences especially at the3′ UTR (Sami-Subbu et al., 1999).

To determine whether the Rp120 shows simi-lar substrate-binding properties, competition reactionswere conducted with 32P-radiolabeled prolamine RNAin the presence of varying amounts of different unla-beled prolamine sequences. In the presence of a 50-fold and 100-fold excess of unlabeled prolamine RNA,the apparent interaction of the Rp120 to labeled pro-lamine RNA was substantially reduced (Figure 2A).In contrast, Rp120 binding to labeled RNA was notaffected in the presence of excess unlabeled prolamineRNA lacking 191 bases of the 3′ end (3′ UTR and thelast 80 nucleotides of the coding region).

To further characterize the binding specificity ofthe Rp120, various segments of the prolamine mRNAwere prepared and used as radiolabeled binding sub-strates. Figure 2B shows that the Rp120 interactsspecifically with the 3′ UTR and not with any otherregion of the prolamine RNA including the poly(A)tail. These results show that the Rp120 interacts withunique sequences or a structural motif located in theprolamine 3′ UTR, which are likely present in glutelinand EF1α RNAs as well.

Rp120 is associated with the cytoskeleton

Developing rice seeds were homogenized in low-ionic-strength buffer containing 1% Triton X-100(cytoskeleton-stabilizing buffer) (Davies et al., 1991;Abe and Davies, 1995) or high-ionic-strength bufferlacking detergent (cytoskeleton-destabilizing buffer)(Davies et al., 1991; Abe and Davies, 1995), andsubjected to low-speed centrifugation to remove largestarch grains as well as nuclei/chromatin. The result-ing supernatants were then fractionated on a 60–80%(low-ionic-strength buffer + 1% Triton X-100) or 50–64% (high-ionic-strength buffer) sucrose density gra-dients. Individual fractions from each of these sucrosegradients were collected, resolved by SDS-PAGE and

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Figure 3. Sedimentation behavior of Rp120 on sucrose density gra-dients under low-ionic-strength (panel A) and high-ionic-strength(panel B) conditions. Developing rice seed extracts were prepared inlow-ionic-strength (cytoskeleton-stabilizing) buffer containing 1%Triton X-100 (A) or high-ionic-strength (cytoskeleton-disruptive)buffer (B) and subjected to sucrose density gradient centrifugation.Various fractions of the gradient were then analyzed by immunoblotusing antibodies to Rp120, the ER integral membrane protein cal-nexin, and actin. Panel A: lane 1, crude extract; lane 2, top solublefraction of the sucrose gradient; lane 3, protein band 1 that sedi-mented just below the soluble protein-60% sucrose interface; lane4, protein band 2 that sedimented just below protein band 1; lane5, protein band collected at the 60–80% sucrose interface. PanelB: lane 1, crude extract; lane 2, top soluble protein fraction ofthe sucrose gradient; lane 3, membrane band at the soluble pro-tein-50% interface; lane 4, membrane band just below the solubleprotein-50% interface; and lane 5, membrane band at the 52–54%interface.

then analyzed by immunoblot with antibodies raisedagainst Rp120, actin and the ER integral membraneprotein calnexin, respectively. Figure 3A shows that,when prepared under low-ionic-strength conditions,the bulk of the Rp120 enters the sucrose gradient andsediments to the dense regions where cytoskeleton el-ements reside as indicated by the presence of actin. Incontrast, the calnexin remained within the soluble (toplayer) phase of the gradient, a location consistent withits solubilization from the ER membrane by 1% Tri-ton X-100. These results indicate that the majority ofRp120 is associated with a dense cellular matrix whichhas sedimentation behavior similar to that displayedby the actin-containing cytoskeleton.

Under high-ionic-strength conditions which desta-bilize the cytoskeleton, a much different protein dis-tribution pattern on the sucrose density gradient wasobserved. Almost all of the Rp120 was located eitherin the soluble fraction or on the lightest sucrose den-sity layer (Figure 2B). Actin was also found mainlyin the lightest fraction of the gradient. Although theER integral membrane protein calnexin was also inthis light fraction, significant amounts were also ob-

Figure 4. Temporal (A) and spatial (B) protein expression patternsof Rp120. Protein extracts were prepared from developing seeds ofvarying age (4 to 12 DAF) or from different tissues and analyzed byimmunoblot with antibodies to Rp120, glutelin or prolamine.

served in denser layers of the gradient. Overall, theseresults showed that the Rp120 is associated with adense cytomatrix in a detergent-resistant but ionic-strength-sensitive manner – properties associated withthe cytoskeleton.

Tissue-specific and temporally specific expression ofRp120

To study the expression pattern of Rp120 in vari-ous tissues, total protein from the roots, leaves ofseedlings, florets and developing seeds were subjectedto immunoblot. When probed with Rp120 antibodies,Rp120 expression was observed only in the develop-ing seeds (Figure 4B). The temporal expression ofRp120 with respect to prolamine and glutelin expres-sion was studied by assessing Rp120 levels in devel-oping seeds of varying age, from 4 to 12 days afterflowering (DAF). Such immunoblot analysis showedthat Rp120 is expressed as early as 4 DAF and attains amaximum steady-state level beginning between 7 and10 DAF and extending throughout seed development(Figure 4A). Hence, the protein accumulation patternof Rp120 differs from that observed for prolamine andglutelin which occurs later in seed development.

Discussion

Non-random distribution of mRNA in the cytoplasmhas been reported in a wide range of eukaryotic sys-tems (Wilhelm and Vale, 1993; St Johnson, 1995;Hesketh, 1996; Singer, 1996). In rice endosperm cells,prolamine RNAs are targeted and accumulated to spe-cific ER subdomains by RNA-dependent process asso-ciated with the cytoskeleton (Li et al., 1993; Muench

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et al., 1998; Choi et al., 2000). Since directed RNAtransport infers the existence of RNA-binding activitythat recognizes prolamine RNAs, we identified andpurified by Poly(U)-Sepharose and heparin-agarosechromatography several RNA-binding proteins froman enriched cytoskeletal-PB fraction. Several RNA-binding activities were identified in the 300 mMPoly(U)-Sepharose elution fraction with approximatemolecular size of 120, 100, 75 and 40 kDa. The40 kDa polypeptide was the main binding activity inthe 1 M KCl fraction that eluted from the Poly(U)-Sepharose column (Sami-Subbu et al., 1999). Subse-quent fractionation of the 300 mM KCl fraction on theheparin-agarose column resolved a single prominent120 kDa band. Antibodies raised against Rp120 wereused to identify the corresponding gene sequences in adeveloping rice seed cDNA library. When the isolatedcDNA sequences were fused in-frame to a hexa-Hiscoding sequence and expressed in E. coli cells, theaffinity-purified protein cross-reacted with the Rp120antibody (data not shown). Thus, the isolated clone en-codes the Rp120 polypeptide. Analysis of the Rp120primary sequence provides some interesting clues onits possible function. Rp120 contains repeated SN,tudor and coiled-coil domains. The SN domains areinvolved in single-stranded nucleic acid binding whilethe coiled-coil domain is a prominent structural motiffor protein-protein interactions. The tudor domain hasbeen suggested to be involved in RNA transport and/orprocessing (Ponting, 1997). For example, mutations inthe tudor containing the homeless gene cause disrup-tions in the transport and localization of specific RNAsduring oogenesis (Gillespie and Berg, 1995), and mu-tations in the tudor domain of SMN reduce ability tointeract with spliceosomal Sm proteins (Raker et al.,1999).

Rp120 is closely related to the transcriptional co-activator, p100, which binds the Epstein-Barr virus nu-clear antigen 2 in a complex with the basal transcrip-tion factors. Despite the close similarity in structure,Rp120 is unlikely to share a similar role in transcrip-tional co-activation as it does not contain any obviousnuclear localization signals. This view is supported byour cell fractionation studies in which Rp120 was notfound in the nuclear fraction (results not shown). LikeRp120, p100 is also able to bind to single-strandednucleic acids, suggesting that it has other functionsbesides activating transcription.

Consistent with the presence of multiple SN do-mains, Rp120 binds RNA. Rp120 interacts withprolamine, glutelin and EF1α RNAs indicating that

Rp120 is likely to be a general RNA-binding protein,which recognizes a wide range of mRNAs expressedduring seed development. Despite this apparent lack ofspecificity toward RNA species, further analysis indi-cated that the Rp120 recognizes specific regions of theprolamine RNA. When the binding reaction of Rp120to labeled prolamine RNA was done in the presence ofexcess unlabeled intact prolamine RNA or truncatedprolamine RNA lacking the 3′ UTR and poly(A) tail,only the intact prolamine RNA efficiently competedthe binding activity (Figure 2A). These results suggestthat the Rp120 binds specifically to sequences or astructural motif in the 3′ UTR. This view was con-firmed by direct binding studies where Rp120 bindsto the 3′ UTR but not 5′ UTR or coding sequencesof prolamine RNA (Figure 2B). Taken together, thebinding properties of Rp120 differ considerably fromthe 40 kDa RNA-binding protein which recognizes theA-rich motif located on either 5′ or 3′ UTR (Sami-Subbu et al., 1999). The coil-coil motif observed atthe C-terminus of Rp120 infers the interaction withother proteins. Indeed, evidence for the associationof Rp120 with the cytoskeleton is suggested. Whenan enriched cytoskeletal-PB fraction, prepared in thepresence of low-salt buffer (cytoskeleton-stabilizing)and non-ionic detergent Triton X-100, was resolvedby sucrose density gradient centrifugation, Rp120sedimented, like actin, to the dense regions of the su-crose gradient. This sedimentation behavior was notdue to the association of Rp120 with membranes ascalnexin, an integral ER membrane marker, was solu-bilized by the detergent and was present in the solubletop fraction of the gradient (Figure 3A). When iso-lated and resolved on sucrose density gradients inhigh-ionic-strength buffers, conditions that disrupt thecytoskeleton (Davies et al., 1991; Abe and Davies,1995), significant quantities of Rp120 were detectedin the soluble fraction of the gradient as well as at thesoluble protein-52% sucrose interface of the gradient(Figure 3B). Hence, under conditions that disrupt thecytoskeleton, Rp120 sediments as a soluble protein oras a complex with a density much lower than that ob-served under conditions that maintain the integrity ofthe cytoskeleton. These distinct sedimentation prop-erties under conditions that stabilize or disrupt thecytoskeleton support the view that Rp120 is associatedwith the cytoskeleton.

Rp120 is expressed only in seeds (Figure 4B) andits temporal protein accumulation patterns indicatethat it is expressed earlier than prolamine and glutelinpolypeptide synthesis during seed development (Fig-

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ure 4A). Previous RNA expression data showed thatglutelin and prolamine RNAs are detected as early as5 DAF (Kim et al., 1993) when Rp120 is synthesized.The temporal pattern of Rp120 expression indicatesthat it plays a role early during seed developmentbefore the onset of storage protein biosynthesis.

In conclusion, the properties of Rp120 are con-sistent with a role in RNA transport or processing.Rp120 binds specifically to the prolamine 3′ UTR,is associated with the cytoskeleton especially thosefound in the PB fraction, and is expressed beforethe onset of storage protein accumulation. Efforts arebeing directed at determining the role of this RNA-binding activity in storage protein RNA localizationand during seed development.

Acknowledgements

R.S-S. is a recipient of a graduate fellowship fromthe Rockefeller Foundation Program in Rice Biotech-nology. This study was supported in part by USDANRICGP Grant 9701420 and WSU CAHE Project0590.

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