Biochimica et Biophysica Acta, 1171 (1992) 88-92 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$115.01)

88

BBAEXP 90416

Short Sequence-Paper

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana A.S.N. R e d d y a,1, A n d r e w J. Czernik b, G y n h e u n g A n c and B.W. Poovaiah a " Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture and Landscape Architecture, Washington State Uniuersity, Pullman, WA (USA), b Department of Molecular and Cellular Neurosciences, Rockefeller Unit,ersity, New York, NY (USA) and c Institute of Biological Chemistry, Washington State University, Pullman, WA (USA) (Received 23 July 1992)

Key words: 70K protein; cDNA; RNA processing; Splicing; U1 snRNP; (A. thaliana)

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant Ul snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant UI snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

In eukaryotic cells pre-messenger RNAs are extensively processed within the nucleus to generate the mature m R N A products. The removal of non-coding intervening sequences, a fundamental process in gene expression in eukaryotes, is one of the nuclear events involved in R N A processing [1,2]. The molecular mechanisms by which cells excise the introns with extremely high precision are an area of intense research [1,3]. Extensive studies with yeast and animal systems have established that uridine-rich small nuclear RNAs (U snRNAs, U1, U2, U3, U4, U5 and U6) together with several proteins that bind to snRNAs play a crucial role in RNA splicing [3,4]. snRNAs and the associated proteins form small nuclear ribonucleoprotein (snRNP) particles [3,4]. Each snRNP contains at least one s n R N A bound to several proteins, some of which are common to all the spliceosomal snRNPs and some are unique to each snRNP [3,4]. The snRNPs bind to p r e - m R N A s and along with other proteins form a large ribonucleoprotein complex called spliceosome where introns are removed and exons are ligated [1,2].

t Current address and correspondence to: A.S.N. Reddy, Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. The nucleotide sequence data reported in this paper will appear in the EMBL/GenBank, DDBJ Nucleotide Sequence Databases under the accession number M93439.

U1 s n R N A is the most abundant of the snRNAs present in eukaryotic cells and is essential for prem R N A splicing [4,5]. U1 snRNP consists of at least 10 polypeptides and a U1 s n R N A which is 165 nucleotides long [3,4,6]. Three of the polypeptides designated as 70K, A and C are unique to U1 snRNP and the other (B,B',D,D',E,F,G) are present in all of the major nucleoplasmic snRNPs [3,4]. Genetic and biochemical analyses have established that the UI snRNP binds to the 5' splice site which is an early event in the splicing pathway [7,8]. Binding of U1 snRNP to 5' splice site involves base pairing between the U1 s n R N A 5' end and the 5' splice site of the p r e - m R N A and this binding appears to be dependent on the presence of U1 snRNP proteins [4,8,9]. The U1 snRNP 70K protein has been shown to bind to the first stem-loop at the 5'end of the U1 s n R N A and is believed to be involved in the 5' splice site recognition a n d / o r U1 snRNP interaction with other snRNP complexes [9,10]. U1 snRNP 70K protein has been cloned and characterized from humans [11-13], Xenopus [14], Drosophila [15] and yeast [16]. U1 snRNP 70K protein is actually 52K in size [12,13,15]. However, it migrates as a 70K protein on SDS gels. Aberrant migration of this protein at 70K in SDS gels is found to be due to highly charged carboxy-terminal half of the protein [12,13]. However, the name 70K protein is retained in the literature [11-16]. To be consistent, we used the same name for the plant homolog.

89 In plants, very little is known about the mechanisms involved in pre-mRNA splicing. U1 snRNA has been isolated from plant systems [17-19]. U1 snRNA gene has been cloned and sequenced from bean [19] and pea [17]. Bean and human U1 snRNA genes are 65% homologous and the predicted secondary structure of bean U1 snRNA is identical to animal U1 snRNA [19]. However, the 70K protein that is specific to U1 snRNP particle has not been characterized from plants. Here we describe, for the first time in plants, the isolation of a c D N A clone that codes for U1 snRNP 70K protein from Arabidopsis which shows a high degree of homology with the human U1 snRNP 70K protein. The plant U1 snRNA 70K c D N A was isolated and identified in the course of screening of Arabidopsis thaliana L. Columbia expression library with affinity10

purified antibodies raised to a synthetic peptide corresponding to amino acid residues 378-402 of the a subunit of the rat brain calcium/calmodulin-dependent protein kinase II. Screening of Arabidopsis Agtll library was performed according to standard procedures [20] using alkaline phosphatase conjugated secondary antibody. Out of the twelve positive clones two clones were identified as U1 snRNP 70K cDNAs by sequencing and were designated as pASNP1 and pASNP2. The synthetic peptide to which antibody was raised showed no homology to deduced amino acid sequences from the cDNA. pASNP1 contained an insert of 1133 nucleotides whereas pASNP2 contained an insert of 775 nucleotides long. The nucleotide sequence of pASNP1 and the deduced amino acid sequence is shown in Fig. 1. pASNP2 nucleotide se-

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A Fig. l. Nucleotide sequence of the Arabidops~ U1 s n R N P 7OK c D N A (pASNP1) clone, c D N A inserts were excised from Agtll clones with EcoRI and subcloned into the multiple cloning site of pBluescript in both orientations. Both strands of the c D N A clones were sequenced by the d i d e o ~ n u c l e o t i d e chain termination method using double stranded plasmid D N A as a template [32]. The deduced amino acid sequence is shown below the nucleotide sequence. Nucleotides are numbered on the top and the amino acids are numbered on the side. The asterisk indicates the beginning of the second c D N A (pASNP2) clone. The nucleotide sequence of the pASNP2 is exactly same as pASNP1 except that it has 16 additional nucleotides ( C T T I ' A A A G T T G T G A T G ) in the 3' untranslated region and the arrow indicates the location of the 16-nucleotide stretch in pASNP2.

90 human [11], Drosophila [15], Xenopus [14] and yeast [16] is shown in Fig. 2. The highest homology was found with the human U 1 snRNP 70K protein followed by Drosophila, Xenopus and yeast• Plant 70K homolog showed about 44% identity and 65% similarity with the human 70K protein. The amino-terminal regions (up to amino acids 197 with respect to human 70K protein, Fig. 2) are highly conserved among the vertebrate, yeast and the plant 70K proteins. Plant 70K protein showed significant homology with the vertebrate 70K protein in the carboxy-terminal also (Fig. 2). However, the carboxy-terminal of the plant 70K protein is not as conserved as amino terminal part. The carboxy-termihal half of the protein is less well conserved among all the known 70K proteins [11-15]. Yeast 70K protein showed the least homology with other plant and animal 70K proteins in the carboxy terminus [16] (Fig. 2). The U1 snRNP 70K protein and other RNA binding domains contain an RNA recognition motif (RRM) of about 90 amino acids long (shown by underscoring in Fig. 2) [5]. Within this RRM, two highly conserved amino acid segments (RNP1 and RNP2) are present [5]. RNP1 is eight amino acids long whereas RNP2 is six amino acids long. The RNP2 consensus is not as

quence is identical to pASNP1 except that it is shorter and contained an extra 16 nucleotides in the 3' untranslated region (Fig. 1). The deduced protein is 303 amino acids long. Computer search of the protein database with the deduced amino acid sequence of pASNP1 using FASTA program indicated that the pASNP1 codes for a plant homolog of the 70K protein. The deduced amino acid sequence showed all the conserved features of other known vertebrate U1 snRNP 70K proteins. These include RNA recognition motif (RRM) with two RNA binding domains (RNP1 and RNP2) and a highly charged carboxy-terminus present in the vertebrate 70K proteins [11-15]• Arabidopsis 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the aminoterminal region as compared to human UI snRNP 70K protein [11]. Hence, the total length of the plant 70K protein could be about 371 amino acids with a size of about 43-45K. In vertebrates the 70K protein size ranges from 52-57K [11,14,15] where as it is 34K in size in yeast [16]. A comparison of the deduced amino acid sequence of the U1 snRNP 70K protein with the published amino acid sequences of U1 snRNP 70K protein from

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Fig. 2. Alignment of the deduced amino acid sequences of the U1 snRNP 70K proteins from Arabidopsis thaliana, human [11,13], Xenopus lael,is [14], Drosophila melanogaster [15] and Saccharomyces ceret,isiae [16]. Alignments were made by using PILEUP program and by visual inspection. Dots represent the gaps inserted to maximize the alignment. The identical and similar amino acids in two or more sequences are boxed. Amino acids are grouped as follows: (A,G) (L,I,V) (F,Y) (H,R,K) (D,E) [16]. The RNA recognition motif (RRM) is underscored and the conserved amino acid regions, RNP1 and RNP2, of the RRM are indicated by dashed lines. A bipartite signal motif for nuclear localization in the plant 7(1K protein is indicated by a double line (NLS) above the aligned sequences [30].

91 conserved as RNP1 consensus. The spacing between the RNP1 and the RNP2 is also conserved. By deletion analysis of the human 70K protein it has been demonstrated that a l 11-amino acid region which contains RRM is essential for binding of 70K protein to U1 snRNA [13]. Crystal structure of RRM of U1 snRNP A protein shows a four-stranded /3 sheet and two a helices and the RNP1 and RNP2 lie side by side on the middle two strands [21]. It is found that the RNA binds to the four-stranded /3 sheet and to the flexible loops on one end [21]. Plant 70K homolog has the RRM and showed very high homology with vertebrate and yeast RRM. The RNP1 and RNP2 are also conserved in the plant 70K protein (Fig. 2). In the 87 amino acid long RRM, 49 (56%) residues are identical between the human and plant U1 snRNP 70K protein. In addition to RNP1 and RNP2, the plant 70K protein contained other amino acids that are conserved in the RRM of all the known RNA binding proteins [5] indicating that it is an RNA binding protein. Carboxy-termini of all the vertebrate 70K proteins have an arginine-rich region and are highly charged with arginine, lysine, histidine, glutamic acid and aspartic acid comprising 53-69% of the amino acids [11,14,15]. The carboxy-terminal half of the plant 70K protein is also rich in arginine and charged amino acids (R,K,H,D,E) constituted 63% of the amino acids. However, yeast 70K protein carboxy terminus is moderately charged (25% R + K + H + D + E) [16]. To investigate the expression of the U1 snRNP 70K gene, RNA from stem, root, leaf and flower was isolated [22] and probed with pASNP1. One band of approx. 1.6 kb hybridized in all the samples (Fig. 3). In humans, Drosophila and yeast the 70K protein is coded by a single gene [12,15,16] whereas in Xenopus it is coded by two or more genes [14]. By disrupting the yeast 70K protein coding gene (SNP1) it has been shown that the 70K protein is essential for the viability of cells [16]. To determine the copy number of the plant UI snRNP 70K protein gene, the total genomic DNA was isolated [23], digested with three different

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restriction enzymes and probed with pASNP1. As shown in Fig. 4, a single hybridizing band was detected in all the lanes and the size of the hybridizing fragment ranged from 4 to 6 kb. Washing of the filters at moderately stringent conditions (2 x SSC, and 0.1% SDS at 55°C for 30 rain) or longer exposure of the blots did not show any additional hybridizing bands. These results indicate that the U1 snRNP 70K protein is coded most likely by a single gene in Arabidopsis. The shorter cDNA (pASNP2) contained 16 additional bases in the 3' untranslated region (Fig. 1). This difference in size could have resulted by alternative splicing. In humans, different transcripts are produced by alternative splicing of a single gene [12]. Whether the difference in the 3' untranslated region has any significance in terms of transcript stability or translational efficiency remains to be explored. The presence of certain elements in the 3' untranslated regions of mRNA has been shown to control the stability of the mRNAs [2]. The conserved nature of the plant 70K protein suggests that the 5' splice site recognition mechanism could be similar between plants and animals. However, structural requirements for intron processing in plants

92

differ from those in vertebrates and yeast [24-27]. In plants, especially in the dicots, high A and U nucleotide content in the introns is necessary for intron processing and plant introns lack pyrimidine tract at 3' end [26,27]. Although the plant introns can be faithfully processed by the animal systems, the vertebrate introns are not processed in plant cells [24,25]. This indicates that plant introns contain some unique elements. Therefore, plants may differ in certain steps of RNA processing as compared to vertebrates [25,26]. The divergence in the carboxy-terminal sequence of the plant 70K protein could contribute to difference in splicing mechanisms between plants and animals. The human 70K protein is a phosphoprotein [4,16,28] and several phosphorylation sites have been found in all the known 70K proteins. We do not know whether plant 70K protein is a phosphoprotein. However, several phosphorylation site consensus sequences [29] for different serine/threonine protein kinases are present within the primary structure, suggesting the plant 70K protein might be a phosphoprotein. U1 snRNP specific proteins move into the nucleus independently of the UI snRNP [3-5]. A bipartite signal motif containing two basic amino acids, a spacer region of ten residues and a basic cluster in which three out of the next five amino acids are basic appears to be required for nuclear localization [30]. The bipartite motif is believed to target the proteins to transport through the nuclear pore [4,6,30]. The plant 70K protein contained a bipartite signal motif (amino acids 208 to 224 shown by double underline in Fig. 2) for nuclear localization. A hydropathy plot of the plant and human 70K proteins are quite similar and both the proteins are highly hydrophilic (data not shown) [11,31]. The availability of cDNA for Arabidopsis U1 snRNP 70K protein should permit further investigations on the role of the 70K protein in splicing in plants using a variety of in vitro approaches and by manipulating its expression in vivo in transgenic plants. Furthermore, the advantages of Arabidopsis for molecular genetic studies and its amenability for transformation should be of great help in our future studies. This work was supported by grants from National Science Foundation (DCB-9104586) and National Aeronautics and Space Administration (NAG100061S/1) to B.W.P. and from Agricultural Experiment Station (Project No. 702) and Plant Biotechnology Laboratory to A.S.N.R. We thank Dr. Gokul Das, Cold Spring Harbor Laboratory for helpful discussions. The software cited is part of the VADMS center, a campus-wide computer resource at Washington State University supported by NIH, WSU's graduate school and WSU's academic computing services. Special thanks to Steve Thompson and Susan Johns of VADMS for their help.

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Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not f...
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