Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize...

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Biochimica et Biophysica Ac

Two novel arginine/serine (SR) proteins in maize are differentially spliced

and utilize non-canonical splice sites

Smriti Guptaa, Bing-Bing Wangb, Gabrielle A. Strykera, Marıa Eugenia Zanettic,

Shailesh K. Lala,TaDepartment of Biological Sciences, Oakland University, Rochester, MI 48309-4401, United States

bDepartment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, United StatescDepartment of Botany and Plant Science-Center for Plant Cell Biology, University of California, Riverside, CA 92521-0001, United States

Received 1 June 2004; received in revised form 28 December 2004; accepted 4 January 2005

Available online 17 March 2005

Abstract

The serine–arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in

both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B.

Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence

similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative

splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at

least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced

transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their

first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively

spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Alternative splicing; Pre-mRNA splicing; EST analysis; Non-canonical splice sites

1. Introduction

The precise recognition and removal of introns from

precursor mRNA is essential for the expression of eukaryotic

genes. However, this process is poorly understood in plants

primarily due to the lack of in vitro extract capable of

efficiently splicing plant introns. Despite sharing basic

structural similarity, recent studies indicate that the mecha-

nisms of intron recognition in plants are likely to be different

from vertebrates and yeast [1–5]. In addition, alternative

splicing, a process by which some eukaryotic genes encode

more than one transcript by differential selection of splice

sites during pre-mRNA have emerged as a major form of

gene regulation in vertebrates. Data generated from the

0167-4781/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbaexp.2005.01.004

T Corresponding author. Tel.: +1 248 270 2875; fax: +1 248 370 4225.

E-mail address: [email protected] (S.K. Lal).

analysis of the human genome indicates that alternative

splicing of more than 75% of the human genes containing

multiple exons dramatically increases the complexity of

proteins [6,7]. Alternative splicing has also been demon-

strated to play a vital role in metabolic processes pertaining to

growth and development [8]. Recent completion of Arabi-

dopsis and rice genome in combination with large amount of

Expressed Sequence Tags (ESTs) from plants suggests that

the regulation of gene expression by alternative splicing in

plants is far more prevalent than initially anticipated [9].

Despite the importance of alternative splicing in plants, the

knowledge of the regulation of this process is rudimentary.

Arginine–serine-rich (SR) proteins are a highly con-

served family of nuclear phospho-proteins that play im-

portant roles in both constitutive and alternative splicing

[10]. These proteins are characterized by the presence of

one or two RNA binding domain (RRM) at the N-terminus

ta 1728 (2005) 105–114

S. Gupta et al. / Biochimica et Biophysica Acta 1728 (2005) 105–114106

and an arginine- and serine-rich region at their C-terminus

[11–13]. They also share a characteristic serine-phosphate

epitope, specifically recognized by monoclonal antibody

mAb104 [14]. The conserved RS domains of SR proteins

through RNA–protein and protein–protein interactions with

other proteins play important roles in the recruitment of other

splicing factors during the assembly of spliceosome. For

example, the simultaneous interaction with the mRNA and

U1-70 K RNP protein by SR protein designated SF2/ASF

plays an important role in the recognition of 5V splice site andis essential for the first cleavage reaction during pre-mRNA

splicing [15]. Similarly, SR proteins (SF2/ASF and SC35)

are involved in bridging the 5V and 3V splice sites by inter-

acting with the U1-70 K protein and U2AF35 [16,17]). Al-

though different SR proteins exhibit differences in splicing

specificity and efficiency with different mRNA substrates,

individual SR proteins can in vitro complement vertebrate

S100 extracts that specifically lack SR proteins [10,13].

Almost every study on SR proteins in plants for the past

decade has been performed on Arabidopsis. Plant SR

proteins share basic structural similarity with the verte-

brates, however they bear characteristic motifs that are not

present in vertebrates. Furthermore, some Arabidopsis SR

proteins appear to bear no vertebrate homologs [5,12,18–

21]. These differences may indicate intrinsic differences

between the splicing machinery of plants and animals. For

example, protein–protein interaction studies using yeast

two-hybrid system indicates that Arabidopsis U1-70 K

interacts with at least four SR proteins. Of these, SR protein

(SR45) does not bear any sequence similarity to vertebrates

and appears unique to plants [21,22]. These observations

suggest that the steps involved during the early spliceosomal

assembly may differ between plants and animals.

The expressions of several SR proteins in plants are

themselves regulated by alternative splicing. The Arabidop-

sis atSRp34/SR1 mRNA undergoes alternative splicing

producing at least five transcripts, of which only one

encodes a full-length protein [23]. The putative protein

products of the other transcripts are variants of their RS

domain. The transient expression analysis of atSRp34/SR1

in protoplast suggests that alternatively spliced isoforms of

atSRp34/SR1 mRNA may not be involved in the regulation

of the transcript levels of the wild type protein. Similarly,

atSRp30 bearing sequence similarity to vertebrate SF2/ASF

is alternatively spliced generating multiple spliced variants

that are differentially expressed in a tissue-specific and

temporal manner [24]. In contrast to atSRp34/SR1, the

overexpression of atSRp30 altered the alternative splicing

pattern of several endogenous genes including atSRp30

mRNA in transgenic plants. Intriguingly, transgenic plants

over-expressing atSRp30 also showed dramatic reduction in

the mRNA levels encoding full-length atSRp34/SR1 protein

and exhibited morphological and developmental abnormal-

ities caused by delayed developmental phase transition [24].

In this report, we describe two novel SR genes in maize,

which we designate zmRSp31A and zmRSp31B. They bear

significant sequence similarity to the Arabidopsis atRSp31

family (atRSp31, atRSp32, atRSp40 and atRSp41) [19,25].

The atRSp31 family represents novel SR proteins in plants

that apparently have no homologs in vertebrates [19,25].

We demonstrate that the expressions of both these genes are

governed by alternative splicing which produces multiple

transcripts. Despite sharing extensive sequence similarity,

the alternatively spliced transcript profiles of zmRSp31A

and zmRSp31B vary dramatically, mostly due to the

differential utilization of splice sites during pre-mRNA

splicing. Our data suggests that some of the alternatively

spliced transcripts are functional to interact with the

ribosomes and translational machinery. We have also

searched the plant genome database, PlantGDB [26], for

homologs of zmRSp31A and zmRSp31B in other plant

species. The sequence analysis indicates that the N-

terminus RNA recognition motifs of these proteins are

highly conserved among different plant species with some

notable variation in their C-terminus containing arginine–

serine-rich region.

2. Materials and methods

2.1. Genomic PCR analysis

Genomic DNA was isolated from maize cultivar B73

leaves using DNAzol reagent (Bethesda Research Labo-

ratory) according to the procedure provided by the

manufacturer. Approximately 1 Ag of genomic DNA was

subjected to PCR amplification using the following primer

sets: 668F (5V-GGCACGAGCATCTCTGAGTC-3V) and

668R (5 V-TGCTATAAATGCTAGCGGCA-3 V) for

zmRSp31A and 770F (5V-GGATTTGTTTGTCGCCAACT-3V) and 770R (5V-CAGGTTTCACTGCATGCAAT-3V) for thezmRSp31B gene. Amplification parameters for zmSRp

genes were optimized using a PCR optimization kit (Opti-

Prime PCR, Stratagene, La Jolla, CA). The spliced align-

ments of the genomic DNAwith their corresponding cDNA

sequence were performed using the GeneSeqer program

[27].

2.2. RNA isolation, Northern and RT-PCR analysis

Total RNA was isolated from maize (B73 inbred) dark

grown roots and shoots using TRIZOL reagent (Invitrogen)

and from 6, 9, 15, 20, 25 and 30 days post pollinated ears as

previously described [28]. 20 Ag of total RNA was resolved

on 1.3% formaldehyde-agarose gels and capillary trans-

ferred to nylon membranes (Nytran). The blot was then

subjected to hybridization by radiolabeled zmSRp cDNA

probes as previously described [29].

The first strand for RT-PCR analysis was synthesized

using oligo dT primers by a commercially available Super

Script II RT-PCR kit from Invitrogen, Inc. Primer sets 668F

and 668R and primer sets 770F and 770R were used for the

S. Gupta et al. / Biochimica et Biophysica Acta 1728 (2005) 105–114 107

RT-PCR analysis of zmRSp31A and zmRSp31B transcripts,

respectively. The resulting PCR products were gel purified

using agarose gel purification GENECLEAN II kit (Bio

101, Inc). The resultant products were cloned using TOPO

TA cloning kit (Invitrogen). DNA sequencing was done by

ABI Prism Dye Terminator sequencing protocol of Applied

Biosystems, Foster City, CA.

Fig. 1. Deduced gene structure and protein sequence of zmRSp31A and zmRS

respectively. The predicted amino acid sequences are shown below the nucleotid

nucleotide and protein sequences. The stop codon is displayed by a *.

2.3. RNA extraction from polyribosomal (PS) complex

Polysomes were extracted from maize root tips following

the protocols previously described [30]. Approximately 1 Alpulverized tissue was suspended in 2 ml of extraction buffer

(0.2 M Tris (pH 9), 0.2 M KCl, 25 mM EGTA, 35 mM

MgCl2, 0.2 M sucrose, 100 mM 2-mercaptoethanol, 50 mg/

p31B: Exons and introns are displayed in upper and lower case letters,

e sequence. The numbers on the right marks the position of their adjacent


ml cyclohexamide, 50 mg/ml chloramphenicol, 1% (v/v)

Triton X-100, 1% (v/v) Brij-35, 1% (v/v) Tween-40, 1% (v/

v) NP40, 1% (v/v) polyethylene 10 tridecyl ether and 1%

sodium deoxycholate). The cell debris was pelleted by 2�centrifugation at 16,000�g for 20 and 10 min, respectively.

The supernatant (SN-16) was loaded on 20–60% exponen-

tial sucrose density gradient by centrifugation at 275,000�g

for 90 min in a swinging rotor SW55Ti (Beckman,

Fullerton, CA). Fractionation was performed using a

gradient fractionator coupled to a UA-5 detector (ISCO,

Lincoln, NE). Non-polysomal (monosomes and mRNP) and

polysomal (2 or more ribosomes per mRNA) fractions were

separately pooled together. Fractions separating the NP and

PS and the fractions containing the bottom gradients were

not pooled and discarded. RNA extraction, as well as

qualitative and quantitative analysis of the RNA from the

combined PS fraction, was performed according to the

procedures described previously [31].

Table 1

SR homologs in different plant species

Plant species Protein

annotation

TUG (ID Number) Protein

length (aa)

Maize ZMRSp31A ZMtuc03-08-11.10205 240

Maize ZMRSp31B ZMtuc03-08-11.9772 239

Arabidopsis ATRSp31 gi:26454661 264

Arabidopsis ATRSp32 At2g46610 250

Arabidopsis ATRSp40 gi:4033468 350

Arabidopsis ATRSp41 gi: 21542447 356

Potato STRSp31A STtuc02-10-23.4522 257

Potato STRSp31B STtuc03-04-26.4624 251

Tomato LERSp31A LEtuc02-10-21.4171 239

Tomato LERSp31B LEtuc02-10-21.13710 251

Sorghum SBRSp31 SBtuc02-10-21.11435 238

Barley HVRSp31A HVtuc02-11-10.5246 254

Barley HVRSp31B HVtuc02-11-10.770 277

Wheat TARSp31A TAtuc03-04-26.31023 204

The table lists the given annotation and the accession number of the TUGs

(Tentative Unique Genes) corresponding to the othologs of SR proteins in

different plants species. The deduced lengths of the SR proteins are derived

from the consensus sequence of their corresponding TUGs.

3. Results and discussion

3.1. Isolation and sequence analysis of genomic and cDNA

clones of zmRSp31A and zmRSp31B

We searched the plant genome database (PlantGDB)

using Arabidopsis and human SR proteins to discover their

putative homologs in different plant species. The results of

this search identified two maize tentative unique genes,

TUGs, ZMtuc03-08-11.10205 and ZMtuc03-08-11.9772,

that showed high degree of sequence similarity to the

Arabidopsis atRSp31 [19]. The ZMtuc03-08-11.10205,

designated zmRSp31A, represented a contig assembly of

16 ESTs derived from 11 distinct maize cDNA libraries,

whereas, ZMtuc03-08-11.9772, designated zmRSp31B,

contig contained 8 ESTs representing 6 different cDNA

libraries. The derived consensus sequence of zmRSp31A and

zmRSp31B contained an open reading frame of 240 and 239

amino acids, respectively, and shared 90% sequence

similarity. Both ZmSRp1 and ZmSRp2 contained two

RRM (RNA Recognition Motif) domains at their N-terminus

and the region rich in arginine and serine at their C-terminus.

Both 5V and 3V UTR of ZmSRp1 and ZmSRp2 shared more

than 80% sequence similarity suggesting their origin from a

recent gene duplication event. Gene specific primers from

the variable 5V and 3V UTR regions of the two genes were

designed and used for PCR amplification on the genomic

DNA isolated from the maize seedlings. The resulting PCR

products were cloned and sequenced. The nucleotide

sequences of zmRSp31A and zmRSp31B were deposited

into the GenBank and assigned accession numbers,

AY616013 and AY616024, respectively. The sequence

alignment of the ZMRSp1 and ZMRSp2 proteins with their

corresponding genomic sequence and the annotation of the

intron/exon boundaries are depicted in Fig. 1. The alignment

depicts the presence of four introns in the coding regions of

both the genes. The positions of the introns are conserved

between the zmRSp31A and zmRSp31B, however, they are

divergent in their length and sequence.

3.2. zmRSp31A and zmRSp31B represent a highly conserved

family of SR proteins in different plant species

We used ZMRSp1 and ZMRSp2 proteins as a query to

search the plant genome database for other homologs in

other plant species. The results of this search identified

putative full-length homologs in potato, tomato, sorghum,

barley and wheat. The homologs from other species ranged

from 204 aa (wheat) to 277 aa (barley) residues in length

and all contained two RRMs at their N-terminus and

arginine/serine-rich region at their C-terminus. The desig-

nated annotation of the genes, accession numbers of their

corresponding tentative unique contig (TUC) and the sizes

of their predicted protein products are displayed in Table 1.

The sequence alignments of the SR proteins listed in Table 1

are displayed in Fig. 2. The high degree of sequence

similarity displayed in the alignment indicates that SR

proteins are highly conserved among different species of

plants. Intriguingly, the degree of sequence similarity among

different species is particularly high at their N-terminus

containing RRM, whereas, the C-terminus containing

arginine/serine-rich regions are divergent in both length

and sequence. Whether these differences in RS domain of

SR proteins of different plant species may contribute to the

splicing difference reported between different plant species

needs further investigation. For example, transgenic tran-

script of maize transposable element Ac is differentially

processed in Arabidopsis and tobacco [32–34]. Further-

more, species barriers in splicing have been reported

between dicots and monocots [35,36].

Fig. 2. Alignment of the deduced protein sequences of the genes displayed in Table 1. The solid area marks the positions at which the same residue occurs in

more than 60% of the sequences, whereas, the shaded area displays the conservative substitutes. The conserved RNP-1 and RNP-2 motifs are enclosed in

rectangles and annotated.


3.3. zmRSp31A and zmRSp31B produce multiple transcripts

by alternative splicing

Northern blot analysis of total RNA extracted from

maize endosperm, root and shoot tissues hybridized to two

major transcripts of approximately 1.4 kb and 1.7 kb and a

minor transcript of 2.0 kb with both zmRSp31A and

zmRSp31B cDNA probes (data not presented). Because

the two cDNA probes shared a high degree of sequence

similarity, which spanned through both their 5V and 3V

UTR, it is highly likely that these probes may have

hybridized to each other’s transcripts during Northern

analysis. The high degree of sequence similarity contrib-

uted to our inability to design gene specific probes despite

repeated efforts. To elucidate the identity of various

hybridizing transcripts on the Northern blot, we subjected

total RNA extracted from maize roots to RT-PCR analysis

using the same primer sets complementary to the 5V and 3VUTR regions of each gene that were used for the

amplification of their genomic sequences. The resulting


RT-PCR products were excised from the gel, cloned and

sequenced. We separately performed the spliced alignment

of the wild type and the alternatively spliced transcripts of

zmRSp31A and zmRSp31B with their corresponding

genomic sequences using the computer software Gene-

Seqer. GeneSeqer contains novel algorithms that perform

spliced alignment of the genomic and their cognate cDNA

sequence and assigns the exon/intron boundaries based on

the strength of the splice site scores [27]. The schematic

representation of the splice alignment of the zmRSp31A

and zmRSp31B genomic DNA with their wild type and

alternatively spliced transcripts are displayed in Fig. 3. As

depicted in panel A of Fig. 3, zmRSp31A produces five

alternatively spliced transcripts, of which four are gen-

erated by the differential selection of the splice sites.

Transcript zmRSp31AI is produced by utilization of

alternative donor and acceptor sites within intron 1 region

results in the creation of two exons and of 31 bp and 340

bp. The overall splicing profiles of some of the alter-

natively spliced transcripts are shared between the two

genes, whereas others are unique to a member. For

example, in transcripts II and III of zmRSp31A and I

and II of zmRSp31B, alternative splicing results in the

inclusion of an exon within the intron 1 sequence.

However, alternative splicing profiles of transcripts I, IV

Fig. 3. Schematic representation of alternative splicing of zmRSp31A and zm

respectively. The positions of the RNP-2 and RNP-1 motifs are indicated. The bl

transcripts, respectively. The positions of the start and stop codons of the wild

alternatively spliced transcripts are marked by stars (*). Alternative donor and acce

non-conanical splice sites of the alternatively spliced transcripts are indicated.

displayed on the right.

and V of zmRSp31A are not detected in zmRSp31B and

III is unique to zmRSp31B.

3.4. Several alternatively spliced transcripts of zmRSp31A

and zmRSp31B utilize non-canonical splice sites

We noted the utilization of several non-canonical splice

sites by the alternatively spliced transcripts of both

zmRSp31A and ZMrsP31B. These non-canonical splice

sites of the alternatively spliced transcripts are displayed in

Figs. 3 and 4. For example, alternative non-canonical

donor (AT) in combination with a non-conical acceptor site

(GA) located 144 bp and 385 bp downstream to the wild

type donor site of intron 1, respectively, creates a cryptic

intron of 242 bp in length within the intron 1 sequence of

transcript zmRSp31AI (Fig. 4A, mRNA I). Similarly, a 48

bp intron separating the two exons created within intron 1

bears a non-canonical dinucleotide AT acceptor site.

Furthermore, the non-canonical alternative spliced donor

site (CT) in combination with the wild type acceptor of the

intron 1 creates a 283 bp intron in this region. The

zmRSp31AII transcript includes a 350 bp additional exon

that is derived from the alternative splice sites utilized

within the intron 1 sequence. This transcript also depicted

a non-canonical splice donor site AA that, in combination

RSp31B. Exon and intron sequences are represented by box and lines,

ack and gray boxes depict exons of the wild type and alternatively spliced

type transcripts are flagged. The positions of the new stop codons of the

ptor splice sites are joined by broken lines. The di-nucleotide sequence at the

The assigned roman numerals to the alternatively spliced transcripts are

Fig. 4. Non-canonical splice sites of the alternatively spliced transcripts of zmRSp31A and zmRSp31B. Exons and introns are represented by upper and lower

case letters, respectively. The donor and acceptor sites are joined by broken arrows. The dinucleotide sequence at the non-canonical splice sites are underlined.

The designated labels of the transcripts are displayed on the left.

Table 2

Non-canonical splice sites of the alternatively spliced transcripts of

zmRSp31A and zmRSp31B

Transcript name Non-canonical splice sites Intron length (bp)

ZmRSp31AI AT–GA 242

ZmRSp31AI GT–AT 48

ZmRSp31AI CT–AG 283

ZmRSp31AII AA–AG 193

ZmRSp31BII GT–TG 100

ZmRSp31BIII GT–TG 343

ZmRSp31BIII TA–AG 571

The table lists the intron length and the di-nucleotide sequence occurring at

the terminal ends of the non-canonical introns of the alternatively spliced

transcripts of zmRSp31A and zmRSp31B genes.


with the wild type acceptor sequence of intron 1, created a

cryptic intron of 193 bp. Similarly, alternative-splicing

events within intron 1 results in the inclusion of 251 bp

exon in zmRSp31AIII transcript; however, no non-canon-

ical splice sites were utilized in this transcript. Transcript

zmRSp31AIV detected only after 38 PCR amplification

cycles (data not presented) was generated by the utilization

of donor site of intron 1 and acceptor site of intron 4.

Exons 2, 3 and 4 were completely missing from this

transcript. Intriguingly, we also discovered a maize EST

(gi: 14701133) with 100% sequence similarity to

zmRSp31A. Apparently the sequence of this EST termi-

nated within the intron 1 sequence. The presence of

polyA+tail indicates that this EST represents a transcript

derived from cleavage and polyadenylation at a promoter-

proximal site within intron 1.

Similar alternative splicing events in the intron 1 region

of zmRSp31B result in the inclusion of 379 bp and 450 bp

exons in zmRSp31BI and zmRSp31BII transcripts, respec-

tively (Fig. 3, panel B). The utilization of non-canonical

acceptor site TG was noted in zmRSp31BII transcript that,

in combination with a cryptic donor site located 150 bp

downstream to the donor site of intron 1, created an intron

of 100 bp in length. Similarly, zmRSp31BIII utilized a non-

canonical acceptor site TG located 21 bp downstream to the

wild type acceptor splice site of intron 2 and non-canonical

donor site TA located 117 bp upstream to the authentic

donor site of intron 4. This transcript was only detected

following 38 cycles of PCR (data not presented).

Virtually all introns contain GT–AG borders with few

exceptions. For example, splice alignment of the available

Arabidopsis ESTs with the complete genome sequence

indicated that approximately only 1.7% of Arabidopsis

introns bear non-canonical splice sites. Furthermore, GC–

AG and AT–AC introns comprised the major fraction of

these non-canonical splice sites [37]. Intriguingly, the

alternatively spliced transcripts of both zmRSp31A and

zmRSp31B frequently utilized non-canonical splice sites.

The list of the 7 non-canonical splice sites discovered in the

alternatively spliced transcripts of zmRSp31A and

zmRSp31B are listed in Table 2. As depicted, none of

these conform to GC–AG or AT–AC splice sites, indicating

that they represent novel non-canonical splice sites. We

have previously reported non-canonical splice sites in maize

but they were associated with either a point mutation of the

wild type splice sites or the heterologous expression of a

gene in transient assays [38–40]. For example, maize mutant

sh2-i, bearing an AG to AA mutation at the terminal end of


an intron of Shrunken-2, gene is recognized as an authentic

splice site in approximately 10% of the transcripts [38].

However, a GT–AT mutation at the donor site of an intron

of Shrunken-2 gene in mutant sh2-7503 completely

abolishes the splice site [39]. The maize transposable

element Ds, when transiently expressed in cell suspensions,

is differentially spliced using non-canonical splice sites [40].

The mechanisms of splicing of the non-canonical splice

sites/introns are largely undetermined. The derived con-

sensus for the donor sites for GC–AG bearing introns are

very similar to GT–AG bearing introns and thus likely

undergo splicing using similar mechanisms as GT–AG

introns [37,41]. The AT–AC bearing introns are spliced by a

minor U12 type spliceosome and recently have been

comprehensively documented in Arabidopsis [42]. The

utilization of these novel non-canonical splice sites in

zmRSp31A and zmRSp31B gene expression may serve as

a model to study the mechanism of their splicing and their

relevance to the regulation of gene expression.

The alternatively spliced mRNAs of the zmRSp31A and

zmRSp31B genes may compete for the splicing factors and

thus may be involved in regulating the levels of the wild

type protein at the post transcriptional levels. For example,

the alternatively spliced mRNA V is prematurely polyade-

nylated within intron 1 of zmRSp31A. The usage of

different polyadenylation sites within the intron has been

recently demonstrated to regulate the expression of FCA

gene in Arabidopsis [43]. Alternatively, these alternative

spliced mRNAs might be translated and participate involved

in functions distinct from the wild type protein including

regulating the levels of wild type protein at the translational

levels.

To identify possible EST evidence of alternative spli-

cing discovered by RT-PCR analysis, we performed the

Fig. 5. Isolation and RT-PCR analysis of polysomal RNA. (A) Sucrose gradient

fractionated on a 20–60% sucrose density gradient. The figure displays the absorba

and polysomes are indicated. (B) EtBr stained gels of the RT-PCR products ampli

zmRSp31A (left panel) and zmRSp31B (right panel). The molecular weight stan

spliced alignment of 395,968 maize ESTs available at the

plant genome database (http://www.plantgdb.org/) against

the genomic sequences of zmRSp31A and zmRSp31B

using the GeneSeqer World Wide Web service (http://

bioinformatics.iastate.edu/cgi-bin/gs.cgi). Intriguingly, of

the 35 maize ESTs that aligned with both the genes, none

corresponded with the alternatively spliced transcripts

determined by experimental analysis (data not presented).

3.5. The predicted translation products of most alternatively

spliced transcripts encode truncated proteins lacking RS

domain

The coding regions of the alternatively spliced transcripts

are often interrupted by in frame stop codons and are

predicted to encode truncated proteins completely lacking

the arginine/serine-rich carboxyl terminus. The positions of

the new stop codons in the alternatively spliced transcripts

are displayed in Fig. 4. The putative translation products of

all the five alternatively spliced isoforms of zmRSp31A

encode truncated forms of the wild type protein. Further-

more, the putative ORF of these entire five transcript only

share exon 1 region with the wild type version of the

zmRSp31A transcript and are thus predicted to encode

protein that will contain only the RNP-2 domain of the

conserved first RRM motif of the wild type protein (Fig. 2).

Similarly, alternatively spliced transcripts zmRSp31BI and

zmRSp31BII are also predicted to encode truncated proteins

of 62 aa and 75 aa residues entailing only the RNP-2

domain of the first RRM motif of the wild type protein.

However, the utilization of two alternative splice sites in

transcript zmRSp31BIII do not result in the frame shift of

the wild protein. The predicted protein product of

zmRSp31BIII would contain the deletion of 7 aa and 39

analysis of polysomes. Crude extracts from the maize root extracts were

nce profile of fractions at 254 nm. The positions of the subunits, monosomes

fied from polysomal and total cellular RNAwith primers complementary to

dards are shown on the right of each panel.

http:\\www.plantgdb.org\

http:\\bioinformatics.iastate.edu\cgi-bin\gs.cgi


aa residues encoded by exon 3 and 4 of the wild type

transcript, respectively. These deletions result in a consid-

erable reduction of the RS domain encoded by this

transcript. This observation contrasts with the alternative

splicing reported for other members of SR proteins in

Arabidopsis that also encode multiple transcripts by

alternative splicing. For example, Arabidopsis atSRp34/

SR1 gene encodes at least five transcripts, of which all are

predicted to encode different variants of their RS domains

while maintaining both their RRM1 and RRM2 domains

[23]. Similarly, alternative splicing of atSRp30 and

atSRp34/SR1 produces three and five transcripts, respec-

tively. The putative proteins of the alternative spliced

transcripts of both these genes produce shorter versions of

their RS domain while maintaining their conserved amino

terminal RRM domain [24]. We note that a single copy U1-

70 K gene in Arabidopsis produces two transcripts, of

which the shorter transcript encodes the wild type protein.

The larger transcript retains an unspliced 910 bp intron and

contains an in-frame stop codon within the retained intron.

Similar to maize genes the retained version of the

Arabidopsis U1-70 K transcript is predicted to encode a

truncated protein containing only the RNP-2 domain of its

RRM motif [44].

3.6. Some alternatively spliced transcripts of zmRSp31A

and zmRSp31B are associated with polysomes

Because the majority of the alternatively spliced tran-

scripts are predicted to encode short protein products

completely lacking the RS and a major portion of their

RRM domain, we investigated the possibility that these

transcript(s) may not be translated and are involved in the

transcriptional regulation of their wild type transcript levels.

Translationally competent (active) mRNAs are able to form

poly-ribosomal complexes by recruiting multiple ribosomes.

We isolated polysomal complexes from cell crude extracts

(SN-16) of maize root tips by fractionation through sucrose-

density gradients. The resultant absorbance profile at 254 nm

is presented in Fig. 5A. RNA was extracted from crude

extract and the pooled polysomal fractions were subjected to

RT-PCR analysis using the same primer sets used during the

RT-PCR analysis of zmRSp31A and zmRSp31B on total

RNA. The ethidium bromide stained resultant PCR products

from the total and polysomal (PS) RNA resolved on 1%

agarose gel are displayed in panel B of Fig. 5. As depicted, in

addition to the major fragment corresponding to the wild

type transcript, two larger transcripts of 1144 bp and 1243 bp

were detected in the PS isolates of zmRSp31A. These

fragments represented the zmRSp31AIII and zmRSp31AII

transcripts, respectively. However, the zmRSp31AI PCR

product of 1408 bp was only detected with the total RNA

isolate. Similarly, one major and one minor product of 915

bp and 1294 were detected in the polysomal RNA with

zmRSp31B specific primers. These represented zmRSp31B

wild type and zmRSp31BI transcripts, respectively. The

zmRSp31BII transcript of 1543 bp was detected only in the

total RNA isolate. These data suggest that some of the

alternatively spliced transcripts are functional to interact with

the ribosomes and translational machinery. The degree of

association of specific mRNAs with polysomes can be used

to determine their translational competence. The differential

gradient centrifugation of the mRNA–protein complex

followed by the examination of the presence/absence of

specific mRNAs in different fractions has been successfully

used to test the ribosomal association of individual mRNAs

[45]. For example, this method in plants has been used to

demonstrate that the differential association of mRNA with

polysomes plays a key role in regulation of gene expression

during dehydration stress induced by water deficit in tobacco

and Arabidopsis [31,46]. Whether alternatively spliced

transcripts of zmRSp31A and zmRSp31B are translated

and their biological relevance in the regulation of gene

expression await further investigation.

Acknowledgements

We thank Drs. Volker Brendel and Julia Bailey-Serres for

their kind support and helpful comments, and Paul McBride

for critically reviewing an earlier version of the manuscript.

The work was supported by Research Excellence Fund,

Oakland University.

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