Elsevier

Gene

Volume 339, 15 September 2004, Pages 25-37
Gene

ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced

https://doi.org/10.1016/j.gene.2004.06.047Get rights and content

Abstract

Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5′ intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5′ splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells.

Introduction

RNA-binding proteins containing repeating arginine and serine residues (SR proteins) are implicated in constitutive and alternative splicing of pre-mRNA (Hastings and Krainer, 2001). A single pre-mRNA can be processed by alternative splicing to produce protein isoforms with different physiological functions. In one extreme case, it has been reported that over 30,000 alternatively spliced products of just one gene, the Dscam gene (115 exons, 95 of which are alternatively spliced), can potentially be expressed in Drosophila (Celotto and Graveley, 2001). Alternative splicing contributes to a diversity of gene products and regulation of gene expression as exemplified by sex determination in Drosophila (Lopez, 1998).

Pre-mRNA splicing requires that a number of factors are organized into the functional entity of the spliceosome. Among SR proteins, a subset of the human ASF/SF2-like proteins plays a unique role in splicing reactions (Kawano et al., 2000). These proteins are involved in the selection and utilization of splice sites by spliceosomes, most likely by interactions with exonic and intronic enhancer sequences (Hastings and Krainer, 2001). Knockouts of the ASF/SF2-like genes in chicken, mouse, Drosophila, or Caenorhabditis elegans do not produce viable phenotypes (Longman et al., 2000). Evidently, the expression of the ASF/SF2-like genes is important for embryogenesis, organogenesis, and morphogenesis.

Alternative splicing has been documented for numerous plant genes (Zhou et al., 2003). Transcription factors regulating diverse biochemical pathways in plants are frequently found to produce alternatively spliced products such as the transcripts of Vp-1 in wheat (McKibbin et al., 2002), the r1 gene in maize (Procissi et al., 2002), and the maize MADS box genes (Montag et al., 1995). The pathogen defense systems of tobacco, Arabidopsis, tomato, and flax rely on alternatively spliced Toll-like receptors for signaling pathogen-elicited responses (Jordan et al., 2002). The systemic wound response pathway of tomato plants involves alternative splicing of prosystemin (Li and Howe, 2001). Plant response to environmental factors such as light or salt concentration, as well as the control of flowering time, require SR-like splicing proteins or alternatively spliced gene products (Mano et al., 1999, Forment et al., 2002, Macknight et al., 2002). The overall importance of alternative splicing in plants still needs to be assessed, but numerous reports suggest that this process is as prevalent in plants as in metazoan organisms (Reddy, 2001).

Of the 18 Arabidopsis SR proteins, atSRp30 and atSRp34 have been identified and characterized as homologues of the human ASF/SF2 alternative splicing factor, revealing 58.1% and 59.4% sequence identity, respectively (Lazar et al., 1995, Lopato et al., 1999). Two additional sequences are annotated as SF2-like in the NCBI database (atSRp34a and atSRp34b) (Lorkovic and Barta, 2002). There are four homologues (SRp30c, SRp55, SRp75, and SRp40) of ASF/SF2 identified and characterized in human cells. By comparison, a search of the C. elegans genome identified seven homologues of human SR genes, one of them being 66% identical to human ASF/SF2 (Longman et al., 2000). Both Arabidopsis genes, like the ASF/SF2-like splicing factor genes from other organisms, produce alternatively spliced mRNA (Lazar and Goodman, 2000).

Although highly homologous, the Arabidopsis genes may not be fully interchangeable with their mammalian counterparts. They neither complement SR protein-depleted HeLa S100 extracts nor bind animal U1-70K protein (Lazar et al., 1995, Lopato et al., 1999). It seems that in plants, some functions of ASF/SF2-like factors, such as bridging 5′ and 3′ splice sites, may be accomplished by yet another category of SR proteins. Indeed, a plant unique SR protein, atSCL33, binds Arabidopsis U1-70K and interacts with a number of plant SR proteins (Golovkin and Reddy, 1999, Lopato et al., 2002). Another plant-specific RS-rich protein, atRSZ33, coexpresses and interacts with atSRp34 in various organs during plant development, with its transcripts being preferentially accumulated in flowers (Lopato et al., 1999, Lopato et al., 2002, Kalyna et al., 2003). Regulated expression of yet another plant-specific SR protein that interacts with the U1-70K factor (SR45) has recently been described in Arabidopsis (Ali et al., 2003).

Families of SR protein-specific kinases such as SRPK or Clk/Sty regulate the function of SR proteins through phosphorylation of their SR domains (Prasad and Manley, 2003). A massive, progressive phosphorylation of ASF/SF2 by SRPK1 is believed to be necessary for protein mobilization to the sites of pre-mRNA splicing and for facilitation of 5′ splice site recognition (Aubol et al., 2003). In plants, the LAMMER family of protein kinases such as, for example, the ethylene-inducible PK12 kinase from tobacco was shown to phosphorylate the Arabidopsis splicing factor atSRp34 (Savaldi-Goldstein et al., 2003). Also, the RSPSK motif was identified in the SR domain of atSRp34. This motif (consensus: K/R–S/T–P–X–K) is a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2 (Lazar et al., 1995). Phosphorylation of SR proteins offers yet another level of regulation of pre-mRNA alternative splicing, in particular, during plant development. The physiological role of LAMMER kinases as well as other kinases is indeed associated with cell cycle or developmental processes in plants (Savaldi-Goldstein et al., 2003).

This paper provides information on three maize ASF/SF2-like genes and their involvement in the selection and utilization of splice sites from different plant pre-mRNA sources in maize cells.

Section snippets

Cloning of zmSRp32 (AY649841)

  • a.

    Genomic polymerase chain reaction (PCR) clone of zmSRp32 from B73: DNA extracted from young leaves by the cetyltrimethylammonium bromide method served as a template for the PCR reaction that contained the zmSRp32 primers (GTCCGCATCCCGTACGTCTCGA and GCATCCAACAATCACCAATGGAAAAG) designed according to the sequence data from the cmst1s.pk007.j15.fis EST clone. This clone was a member of the PCL279954 cluster of 49 ESTs with homology to the atSRp34 cDNA. The Advantage Genomic PCR kit (BD Biosciences

There are at least three ASF/SF2-like genes in maize

A comparison of genomic DNA and full-length cDNA of zmSRp30, zmSRp31, and zmSRp32 indicated similar intron/exon architecture (Fig. 1). Twelve introns were identified for these particular cDNA clones in addition to one intron within the 5′ UTR. The first eight exons encoded two highly conserved RNA binding domains (RBDs). Exons 5 and 6 contained the SWQDLKD signature sequence found in all ASF/SF2-like proteins (Fig. 2A), and exon 4 encoded a glycine hinge connecting two RBD domains. This domain

Discussion

We report here on the identification and characterization of ASF/SF2-like alternative splicing factors in maize. Like the human ASF/SF2 homologue, maize genes contain two RBDs, conserved RNA recognition elements RNP-1 and RNP-2, the SWQDLK sequence signature of the ASF/SF2 factors, and the SR domain. Three ASF/SF2-like splicing factors are described here; however, a possibility exists that there are still more maize homologues of ASF/SF2. Since four ASF/SF2-like genomic sequences have been

Acknowledgements

The authors would like to thank Evgueni Ananiev for kindly providing the BAC clones and genomic DNA from the oat–maize addition lines, and for his critical evaluation of the manuscript. A Discovery grant from Pioneer Hi-Bred International (a DuPont company) supported this work.

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