A nuclear-encoded chloroplast RNP-80 protein from bean binds to a thymine-rich sequence of single-stranded DNA

Plant Science 1I I (1995)199-207

A nuclear-encoded chloroplast RNP-80 protein from bean binds to a thymine-rich sequence of single-stranded DNA * Yasushi Kawagoea, Eric C. Achbergerb, Sue G. Bartlettc, Norimoto Murai*“‘” ‘Departments

of Plant Pathology and Crop Physiology. Louisiana State University and LSV Louisiana ‘Microbiology.

‘Biochemistry.

Louisiana State University.

Louisiana State University and LSV

Agricultural

Center, Baton Rouge,

70803- 1720, USA Baton Rouge, Louisiana 70803, USA

Agricultural

Center, Baton Rouge, Louisiana 1720. USA

Received 22 May 1995; revision received I7 July 1995; accepted 4 August 1995

A cDNA encoding a chloroplast homolog of RNP-80, designated bean-RNPI , has been isolated from a library constructed from bean cotyledon RNA. The protein contains a chloroplast transit peptide sequence, an acidic domain, and two RNP-80 domains. Gel mobility shift assays with a purikd fusion proteins from Escherichia coli demonstrated that the protein binds specifically to thymine-rich single-stranded DNA. The protein interaction with double-stranded DNA was not detectable. Sl nuclease footprinting assays demonstrated that the protein protects a 21-nucleotide thymine-rich sequence from Sl nuclease digestion. In addition, hypersensitive sites were generated upstream of the binding site. Implications of these findings for the possible in vivo function of bean RNPl are discussed. Keywords: Bean; Footprinting;

Gel-shift assay; RNP-80; Single-stranded

DNA T-rich sequence

1. Introduction

quence, designated RNP-80 or RNP consensus RNA-binding domain (CS-RBD) [l-4]. The three

Several families of RNA-binding proteins have been identified based on common sequence motifs implicated in RNA binding. One class of RNAbinding proteins contains one or more copies of a characteristic ribonucleoprotein consensus se-

dimensional structure of the RNP-80 motif of Ul A protein was independently determined by X-ray crystallography [5] and NMR [6]. The RNP-80 motif contains four &strands which form an antiparallel B sheet that packs against two a-helices in a compact folded structure (&x&343). Other proteins with RNP-80 domains appear to share a similar tertiary structure. However, the elucidation of RNP-80 structure was not sufftcient to understand the mechanism by which RNP-80 proteins

*Accession number X82030 at the EMBL Nucleotide Sequence Database. * Corresponding author, Tel.: (+I-504) 388 1380; Fax: (+I%%) 388 1415.

0168~9452/‘95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04216-M

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select their respective target RNAs. Thus, a membership in the RNP-80 family is not of itself predictive of the mode by which the protein binds to RNA [2,4]. Genes for nuclear-encoded chloroplast RNP-80 proteins have been cloned by four different methods. These involved screening cDNA libraries with degenerate oligonucleotide probes deduced from N-terminal amino acid sequences [7,8], screening cDNA expression libraries with an antibody raised against a mRNA 3 ‘-end binding protein [9], screening with double-stranded DNA probes [lO,ll], and PCR amplification with degenerate primers 1121. Common features shared among these proteins include the presence of an Nterminal chloroplast transit peptide and a small acidic domain followed by two copies of the RNP80 domain. Five nuclear encoded RNP-80 genes have been isolated from tobacco, suggesting multiple roles of RNP-80 proteins in plastids [7,8]. A phylogenetic tree constructed based on sequence similarities suggested that the diversity of the five tobacco RNP-80 proteins was generated through a series of gene duplications [8]. The function(s) of chloroplast RNP-80 proteins in plastids is not well understood. However, depletion of the spinach 28RNP together with several other proteins from chloroplast extracts inhibited mRNA 3’-end processing, suggesting that the 28RNP may play a role in the 3’-end processing [9]. RNP-80 proteins are also believed to be involved in intron splicing and mRNA stability [7,9]. Although evidence indicates that chloroplast RNP-80 proteins bind to nucleic acids [ 10, 1 1,131, little is known about binding specificities of each RNP-80 protein. The spinach 28RNP was reported to recognize structural components that are common to some mRNA 3 ‘-ends rather than primary sequence [9]. In contrast, three tobacco RNP-80 proteins, cp28, cp31, and cp33, were shown to bind preferentially poly(U) and poly(G) RNA and to have higher binding affinities for double-stranded DNA than for the single-stranded DNA at 0.1 M NaCl [13]. However, these studies did not identify the target sequence of chloroplast RNP-80 proteins. We are interested in understanding the molecular nature of DNA-binding proteins that

Science 111 (1995) 199-207

recognizes the proximal promoter of the bean seed storage protein phaseolin gene [14]. One of the prominent DNA-binding proteins, AG-1, binds to a AT-rich sequence, AAAAAG(AlG)CAA, located at three separate sites (-376/-367, -356/-347, -191/-182) in the proximal promoter. To isolate cDNA for AG-1 we screened a bean cDNA library by cotyledon expression southwestern analysis using a double-stranded DNA probe consisting of AG-l-binding sites. However, positive clones isolated were found to be products of a nuclear-encoded chloroplast RNP80 gene, designated bean-RNPI. To understand the interaction between bean-RNPl and the AG- 1 probe, we prepared a fusion protein consisting of bean-RNPl and the Escherichia coli maltose binding protein. Gel mobility shift and Sl nuclease protection assays with the purified fusion protein demonstrated that the protein binds specifically to thymine-rich single-stranded DNA. The results define the target sequence of the bean RNP-80 protein and thus should shed light on the potential in vivo function of this protein in plastids. Furthermore, the results also provide a basis for designing new DNA probes to isolate cDNAs of the AG-1 protein. 2. Materials and methods 2.1. cDNA expression library construction Total RNA was isolated from developing cotyledons of Phaseolus vulgaris L. cultivar Contender by a modified hot-phenol method [ 15,161. Poly(A)+ RNA was purified with an oligo-(dT) cellulose column (Stratagene). A cDNA library was constructed with the ZAP-cDNA Synthesis kit (Stratagene) according to the manufacturer’s directions. The primary cDNA library consisting of 1.0 x 10’ plaque forming units was amplified and stored at 4°C. 2.2. Screening

cDNA expression oligodeox) ribonucleotides

library

with

Cloning of a putative DNA binding protein from the cDNA library was conducted according to standard protocols [17] with the following modifications. Binding buffer consisted of 12 mM Tris-HCl (pH 7.7), 5 mM MgClt, 60 mM KCl, 20

Y. Kawagoe el al. /Plant Science Ill (1995) 199-207

mM HEPES (pH 8.4). Two oligodeoxyribonucleotides containing a putative AG-1 binding site [14], TGCGCAAGAAAAAGACAAAGAACAAAGAAAAAAGACAAA and CTGTTTTGTCTTTl-fTCTTTGTTCTTTGTCTTI-IT C, were annealed and 3’-end labeled with [cr32P]dATP and [032P]dTTP using Klenow fragment. The DNA probe (1.9 x lO’counts/min) and sonicated calf thymus DNA (250 mg) were added to the binding buffer (40 ml) to screen 3 x lo5 p.f.u. Plasmids were generated from positive clones by in vivo excision according to the manufacturer’s directions (Stratagene). Single-stranded DNA was isolated and used as template for sequencing reactions using Sequenase (USB). A set of 5’-deletion mutants was generated with exonuclease III and mung bean nuclease according to the manufacturer’s directions (Stratagene). 2.3. Maltose binding protein (MBP)-bean-RNP fusion protein from E. coli The 1.25kb XhoI fragment was inserted into the (New England BioLabs). The MBP-bean-RNP and MBP-&gal-o, which is encoded in the Pmal-CR1 vector, were expressed in E. coli strain XLl-Blue (Stratagene) and purified from cytoplasmic proteins according to the manufacturer’s directions. Concentrations of purified proteins were determined by the Bradford method (Bio-Rad) using bovine serum albumin as a standard. The purified proteins were stored in a buffer consisting of 10 mM sodium phosphate (pH 7.2), 0.5 M NaCl, 1 mM sodium azide, 10 mM & mercaptoethanol, 1 mM EGTA, and 10 mM maltose at 4°C. Safl site of pMal-cRI

2.4. Gel mobility shifr assays with the purifiedfusion protein A NszIIDral fragment (-3911-228) of the phaseolin promoter containing the two AG-1 binding sites was cloned into the PstI and SmaI sites of pUC18. Three types of 116-bp DNA probes were generated by 3’-end labeling with Klenow fragment at the Hind111 and/or Bcfi sites. DNA probes were purified from 8% (w/v) nondenaturing polyacrylamide gel. Gel mobility shift assays were performed as described [14] with the following modifications. Final incubation buffer contained 8

201

mM Tris-HCl (pH 7.7), 13 mM HEPES (pH 8.4), 8% (v/v) glycerol, 100 mM NaCl, 0.05 mg/ml poly(dI)-poly(dC) (Pharmacia), 1 x lo4 counts/mm DNA probe (approximately 10 fmole), and purified proteins. After 30 min incubation, reaction mixtures were loaded on 7% (w/v) polyacrylamide gels. Gels were dried on 3MM paper and exposed to X-ray films for l-3 days. 2.5. Sl nuclease footprinting assay The DNA probe was 3 ‘-end labeled at the HindII1 site as described above. Single-stranded DNA probe was generated after boiling and quick cooling on ice. The probe was used without further purification. The DNA probe (1 x lo5 counts/ min) was mixed with proteins and incubated for 30 min in 30 ml of the binding buffer. Then 90 ml of Sl nuclease buffer consisting of 100 mM NaCl, 50 mM sodium acetate (pH 4.5), 4.5 mM ZnS04, 20 mg/ml ssDNA (Salmon testes DNA, Sigma), and 22.2 units/ml Sl nuclease (Pharmacia) were added to the reaction mixtures. After a further IO-min digestion at 25”C, the reaction was stopped by adding 30 ml stop solution consisting of 4 M ammonium acetate, 50 mM EDTA, and 50 mgml tRNA, followed by phenol/chloroform and subsequent chloroform extraction. After ethanol precipitation, the DNA fragments were dissolved in a gel loading solution consisting of 40% (v/v) 8 mM EDTA, 0.02% (w/v) formamide, bromophenol blue, and 0.02% (w/v) xylene cyanol. DNA fragments were separated in an 8% (w/v) sequencing gel. DNA sequence ladders were obtained from pBluescript SK(-) ssDNA with a reverse primer and used as size markers. 3. ReauIts Seven positive clones were isolated after primary screening of the cDNA expression library from bean cotyledon RNA. A single clone contained 0.94 kb, two clones contained 1.13 kb, and four clones contained 1.25-kb DNA inserts, each representing different 5’-ends of the identical DNA sequence. The DNA sequence of the fulllength 1.25-kb fragment is shown in Fig. 1. Translation of the cDNA sequence into a putative protein sequence revealed an open reading frame

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Science I II (19951 199-207

CTCGAGTTTCATCATCACMC

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781 CIX: CTT (3% Gee lTG AAT GAT GTG GAA CTA GAAGGA aa; ALGALNDVER 841 GAA GG4 AAA CGT GCA CAA ca: F G K R A 0 G 906 976 1047 1118 1189

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Fig. I. Nucleotide and deduced amino acid sequences of bean-RNPI. The amino acid sequence marked by bold letters denotes the putative chloroplast transit peptide. A conserved cleavage site motif ISA-A [22] is underlined. Acidic amino acids in the N-terminus of the mature protein are indicated by asterisks. Two RNP-80 motifs are underlined.

Y. Kawagoe et al. /Plant Science 111 (1995) 199-207

RNP-80 ball-RNPl Tcbacco

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Fig. 2. A comparison of ammo acid sequences of two RNP-80 motifs encoded by bean-RNPl and four related genes. The amino acid sequences encoded by tobacco cp33 [7], spinach 28RNP [9], maize NBP (1 I], and Arabidopsis RNP-T [IO] were compared to the sequence of bean-RNPI (this study). Two RNP-80 motifs are boxed. Gaps were not introduced to optimize sequence alignment in the RNP-80 motifs and short linkers connecting the two RNP-80 motifs. Dashes denote amino acid identity with bean-RNPI. Conserved amino acids shared among the five proteins are indicated by asterisks below the sequence.

(ORF) of 287 amino acid residues with an estimated molecular size of 31.2 kDa. The two truncated cDNAs, 1.13 kb and 0.94 kb, start with Leu (33rd residue) and Glu (98th residue), respectively. The N-terminal region of about 70 amino acid residues is rich in serine and has a net positive of charge, showing properties characteristic chloroplast transit peptides [ 181. A conserved cleavage site motif, ISA-A [19], is found from amino acid residues 69-72. The putative cleavage site is followed by a sequence rich in acidic amino acids. A search of the EMBL database indicated that the putative ORF shares sequence similarities with chloroplast RNP-80 proteins. These include the five chloroplast RNPs of Nicoriuna sylvestris [7,8], the 28RNP of Spinacia oleracea 191,and the RNPT of Arabidopsis fhaliana [lo]. The putative nucleic acid binding protein was thus designated beanRNPl. The conserved RNP-80 motif regions of bean-RNPl was compared to those of four RNP80 proteins from distantly related plants: tobacco

cp33 171; spinach 28RNP [9]; maize NBP [I I]; Arabidopsis RNP-T [lo] (Fig. 2). Spinach 28RNP, maize NBP, and Arabidopsis RNP-T share 79-82% amino acid identities in the regions, whereas bean RNPl and tobacco cp33 have 46-48% and 42-46% identities, respectively, to the former three proteins. The results suggest that bean-RNPl represents a new type of chloroplast RNP-80 protein. Duplication of RNP-80 protein genes seems to have occurred at least twice, generating three types of genes for RNP-80, which are represented by bean-RNPl, tobacco cp33, and the three genes for RNP-80 from maize, spinach, and Arabidopsis. In addition, strong sequence identities among maize NBP (monocot), spinach 28RNP (dicot), and Arabidopsis RNP-T (dicot) imply that the two gene duplications took place long before the divergence of monocots and dicots. The 287 amino acid residues of bean-RNPI plus seven amino acid residues at the N-terminus were fused downstream of the maltose binding protein (MBP). The 74-kDa MBP-bean-RNP fusion pro-

204

Y. Kawagoe

et al. /Plant

A

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Fig. 3. Gel mobility shift assays using single- and doublestranded DNA. (A) Strand-specific 3’-end labeling. The 116bp fragment derived from the &phaseolin promoter [IS] is flanked by Hind111and &/I sites. The T-rich strand was 3’-end labeled with 3zP at the WI site (lanes I and 2) prior to digestion with HindHI, and the A-rich strand was 3’-end labeled at the HindlII site (lanes 3 and 4) prior to digestion with &/I. Both strands were labeled at Hind111 and &/I sites (lane 5). Heat denotes the incubation of respective DNA probes in boiling water for three min prior to electrophoresis on a nondenaturing 7% (w/v) polyacrylamide gel. (B) Strand-specific binding of bean-RNPI. Both strands of the I l6-bp fragment were 3’-end labeled with 32P. Double-stranded DNA (lanes l-3) and separated single-stranded DNA (lanes 4-11) were mixed with MBP-&gal-a (lanes 2.5.6) or MBP-bean-RNP (lanes 3, 7-l I). No purified protein was added to samples in lanes I and 4. The incubation mixtures were analyzed by electrophoresis on a 7% (w/v) nondenaturing polyacrylamide gel.

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tein and 52-kDa MBP-P-gal-o control protein were expressed in E. coli and purified from cytoplasmic proteins using an amylose resin column. Gel mobility shift assays were performed to determine relative affinities of bean-RNPl for single versus double-stranded DNA using a 116bp DNA fragment derived from the P-phaseolin promoter [14]. The preliminary results showed that two single-stranded DNAs generated by heatdenaturation treatment migrated differently in a nondenaturing polyacrylamide gel. To distinguish two strands, we conducted strand-specific 3’-end labeling with 32P.The thymine-rich strand labeled at the Hind111 site migrated slower than the adenine-rich strand labeled at the BcII site (Fig. 3A). The control MBP-P-gal-o protein did not show any detectable binding to either the doubleor single-stranded DNA probe (Fig. 3B, lanes 2, 5, and 6). In contrast, the MBP-bean-RNP fusion protein bound specifically to the T-rich singlestranded DNA, and the DNA/protein complex remained at the origin (Fig. 3B, lanes 7-l 1). The results from titration assay suggest either cooperative binding of the fusion proteins to the DNA, protein-protein interactions, or both. S 1 nuclease footprint assays were used to define specific protein binding sites in a single-stranded DNA. To our knowledge, this is the first such example of Sl nuclease footprint assays. Since the target DNA does not possess apparent inverted repeats, protection should arise from authentic protein/DNA interaction. Although digested fragments were not evenly distributed along the DNA after limited digestion of DNA with Sl nuclease, DNA fragment ladders were obtained in a reproducible manner. Control MBP-@gal-o protein had no appreciable effect on the ladder of DNA fragments generated by Sl nuclease (Fig. 4, lanes 2 and 3). In contrast, the addition of MBPbean-RNP generated the distinguishable pattern of bands compared with MBP-&gal-o (Fig. 4, lanes 3 and 4). First, a strong protection occurred at a 15nucleotide thymine-rich sequence, TTGTCTTTTTCTTGC, corresponding to the 5 ‘end AG-1 binding site (-3671-377) of the phaseolin promoter. This target sequence was a part of the oligodeoxyribonucleotide probe used

Y. Kawagoe et al. /Plant Science 111 (1995) 199-207

lnta-t

DNA

(516 nt)

probe

St rung hypersensitive

sit

Weak hypersensitive

sit

/g z

Weak protection

2

for cDNA library screening and gel mobility-shift assay. A weak protection was also found with the 5 ‘-flanking region, TG’ITCT. Second, hypersensitive sites were generated upstream of the protection site. Weak and strong hypersensitive sites started from 14 nucleotides and approximately 33 nucleotides upstream of the 5 ‘-end of the weak protection site, respectively. The results suggest two possibilities for generation of hypersensitive sites. Dne is that the fusion protein interacts with thymine-rich sequence, altering DNA conliguration in such a way that the upstream region became more susceptible to Sl nuclease attacks. The second possibility is that Sl nuclease interacted with the DNA-bound fusion protein, and the protein-protein interaction in turn sequestered the Sl nuclease to the site identified by the hypersensitive sites. The two possibilities are not mutually exclusive and further discussed later. 4. IXXussien We have cloned a nuclear-encoded chloroplast RNP-80 protein gene, designated beun-RNPl, from a bean cotyledon cDNA expression library. Arubidopsis RNP-T and maize NBP, both of which contain chloroplast transit peptide sequences and two copies of the RNP-80 motif, were isolated by similar methods with different DNA probes [lO,ll]. However, the molecular basis for interactions of RNP-T and NBP with respective DNA probes has not yet been reported. Here, we demonstrated that bean RNP-80 protein preferentially binds to the thymine-rich sequence of singlestranded DNA. It is likely that bean-RNPl binds to single-stranded RNAs in vivo. However, since binding studies with single-stranded RNA have been difficult to perform, we used instead singlestranded DNA in this study. Results of the Sl nuclease footprinting assay provided insights into the interaction between bean-RNPI and the target DNA. First, the interaction is sequence-specific singleand strandedness is not sufficient for the interaction. Second, the target sequence is a thymine-rich sequence. Fourteen out of 21 bases are thymine residues and the longest stretch of thymine residues is five. It remains to be determined

:B Strong protection

I23.J Fig. 4. Sl nuckase footprinting assay. The T-rich strand was 3’cnd labeled and mixed with no protein (lane 2), 3 pm01 of purified MBP-&gal-a (lane 3). or 0.75 pmol of purified MBPbean-RNP (lane 4). A T-sequencing reaction of pBIuescript SK(-) ssDNA (Stratagene) with a reverse primer was used to serve as size markers. Strong protection ¬es the region where differences in band intensities between lanes 3 and 4 are more than fivefold. The sequence protected weakly from Sl nuclease digestion is also indicated. Also shown on the right are regions where strong and weak hypersensitive digestion were ObSClVCd.

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Y. Kawagoe YI al. /Plant

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whether a long stretch of thymine (>5) or thymine-rich sequence in general is necessary and sufftcient for the interaction. Third, the binding does not require secondary structures such as stem-loops. If the interactions involved a stem structure, two or more separated regions would be protected from Sl nuclease digestion because stems are expected to form double-helical structure. Finally, since the binding is sequencespecific, it is likely that the protein recognizes bases. However, this does not exclude the possibility that the protein interacts with the DNA backbone as well. Although we have not compared relative affinities of the protein for single-stranded DNA versus the corresponding RNA, it is likely that absence of 2’-hydroxyl group in deoxyribose and presence of 5-methyl group in thymine may not interfere with the binding reaction. Existence of poly(U) binding proteins in the chloroplast has been demonstrated by several groups. Nickelsen and Link [20) detected proteins of 58, 62, and 70 kDa that bind uridine-rich sequences in mustard chloroplast extracts in a Mg’+-dependent manner. Since our binding solution did not include Mg2+ or other divalent cations and since mature bean-RNPl is much smaller (25 kDa) than the mustard proteins, the bean-RNPl is not likely to be a counterpart of the mustard poly(U) binding proteins. Li and Sugiura [13] demonstrated that three tobacco chloroplast RNP-80 proteins, cp28, cp31, and cp33. bind to homopolymer poly(U) and poly(G), although the relative affinities of the three proteins for doublestranded calf thymus DNA were estimated to be greater than those for single-stranded DNA at 0.1 M NaCl. Bean-RNPl favors single-stranded DNA in reactions containing 0.1 M NaCl. The reason for this discrepancy is not clear. However, it is conceivable that Li and Sugiura’s dsDNAcellulose contained regional strand openings during the binding reactions, UV-crosslinking experiments found several proteins in spinach chloroplast extracts that interact with the 3 ’ region of petD mRNA containing uridine-rich sequence (19,211.

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ment. This work was supported in part by a grant from the Louisiana Education Quality Support Fund (199 l -94)-RD-B7 to NM. References 111 C.G.

Dreyfuss, Conserved structures and RNA-binding proteins. Science,

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121 G.D. Dreyfuss, M.J. Matunis, S. Pinol-Roma and C.G Burd. hnRNP proteins and the biogenesis of mRNA. Annu. Rev. B&hem.,

I31

D.J. Kenan, CC. tion:

towards

identifying

Trends B&hem.

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62 (1993) 289-321. determinants

We thank Biology

members of the Plant Molecular Laboratory for support and encourage-

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Sci., I6 (1991) 214-220.

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recognition: a family matter? Cell, 73

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ISI

K. Nagai,

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Jessen, J. Li and P.R.

Evans, Crystal structure of the RNA-binding the UI

small nuclear ribonuckoprotein

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