Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes

BackgroundSeveral recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes.ResultsIn a comprehensive analysis of the Arabidopsis thaliana genome, we identified 6,772 introns that exhibit a NAGNAG acceptor motif. Alternative splicing at these acceptors was assessed using available EST data, complemented by a sequence-based prediction method. Of the 36 identified introns within 30 SR and SR-related protein-coding genes that have a NAGNAG acceptor, we selected 15 candidates for an experimental analysis of alternative splicing under several conditions. We provide experimental evidence for 8 of these candidates being alternatively spliced. Quantifying the ratio of NAGNAG-derived splice variants under several conditions, we found organ-specific splicing ratios in adult plants and changes in seedlings of different ages. Splicing ratio changes were observed in response to heat shock and most strikingly, cold shock. Interestingly, the patterns of differential splicing ratios are similar for all analyzed genes.ConclusionNAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. A lack of extensive EST coverage can be compensated by using the proposed sequence-based method to predict alternative splicing at these acceptors. Our findings indicate that the differential effects on NAGNAG alternative splicing in SR and SR-related genes are organ- and condition-specific rather than gene-specific.

[1]  Volker Brendel,et al.  The ASRG database: identification and survey of Arabidopsis thaliana genes involved in pre-mRNA splicing , 2004, Genome Biology.

[2]  S. Stamm,et al.  Function of Alternative Splicing , 2004 .

[3]  W. Filipowicz,et al.  Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery , 1996, Plant Molecular Biology.

[4]  W. Filipowicz,et al.  Pre-mRNA splicing in higher plants. , 2000, Trends in plant science.

[5]  A. Reddy,et al.  An SC35-like Protein and a Novel Serine/Arginine-rich Protein Interact with Arabidopsis U1-70K Protein* , 1999, The Journal of Biological Chemistry.

[6]  Michael Hiller,et al.  Widespread and subtle: alternative splicing at short-distance tandem sites. , 2008, Trends in genetics : TIG.

[7]  H. Goodman,et al.  Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Tetsuya Sakurai,et al.  RARGE: a large-scale database of RIKEN Arabidopsis resources ranging from transcriptome to phenome , 2004, Nucleic Acids Res..

[9]  S. Brenner,et al.  Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements , 2007, Nature.

[10]  Yimeng Dou,et al.  Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site. , 2006, RNA.

[11]  Lei Li,et al.  Alternative splicing of prosystemin pre-mRNA produces two isoforms that are active as signals in the wound response pathway , 2001, Plant Molecular Biology.

[12]  Stephen M. Mount,et al.  Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis , 2006, BMC Genomics.

[13]  D. Church,et al.  Spidey: a tool for mRNA-to-genomic alignments. , 2001, Genome research.

[14]  G. S. Ali,et al.  ATP, phosphorylation and transcription regulate the mobility of plant splicing factors , 2006, Journal of Cell Science.

[15]  J. Manley,et al.  SR proteins and splicing control. , 1996, Genes & development.

[16]  Anireddy S. N. Reddy,et al.  Nuclear Pre-mRNA Splicing in Plants , 2001 .

[17]  R. Ophir,et al.  Intron retention is a major phenomenon in alternative splicing in Arabidopsis. , 2004, The Plant journal : for cell and molecular biology.

[18]  A. Barta,et al.  Ectopic expression of atRSZ33 reveals its function in splicing and causes pleiotropic changes in development. , 2003, Molecular biology of the cell.

[19]  Yael Mandel-Gutfreund,et al.  Alternative splicing regulation at tandem 3′ splice sites , 2006, Nucleic acids research.

[20]  Rolf Backofen,et al.  Assessing the fraction of short-distance tandem splice sites under purifying selection. , 2008, RNA.

[21]  E. Birney,et al.  Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. , 1993, Nucleic acids research.

[22]  D. Black.  Mechanisms of alternative pre-messenger RNA splicing. , 2003, Annual review of biochemistry.

[23]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[24]  B. Graveley.  Sorting out the complexity of SR protein functions. , 2000, RNA.

[25]  P. Ferranti,et al.  Alternative Nonallelic Deletion Is Constitutive of Ruminant αs1-Casein , 1999, Journal of protein chemistry.

[26]  K. Shimamoto,et al.  The Serine/Arginine-Rich Protein Family in Rice Plays Important Roles in Constitutive and Alternative Splicing of Pre-mRNA[W] , 2005, The Plant Cell Online.

[27]  V. Brendel,et al.  Genomewide comparative analysis of alternative splicing in plants. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Anireddy S N Reddy,et al.  Plant serine/arginine-rich proteins and their role in pre-mRNA splicing. , 2004, Trends in plant science.

[29]  B. Rogina,et al.  The chicken homeobox gene Hoxd-11 encodes two alternatively spliced RNA species. , 1995, Biochemistry and molecular biology international.

[30]  S. Brenner,et al.  The evolving roles of alternative splicing. , 2004, Current opinion in structural biology.

[31]  A. Reddy.  Alternative splicing of pre-messenger RNAs in plants in the genomic era. , 2007, Annual review of plant biology.

[32]  G. Ast,et al.  Different levels of alternative splicing among eukaryotes , 2006, Nucleic acids research.

[33]  Thangavel Alphonse Thanaraj.  A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures , 1999, Nucleic Acids Res..

[34]  David L. Smith,et al.  Characterization of alternative oxidase (AOX) gene expression in response to methyl salicylate and methyl jasmonate pre-treatment and low temperature in tomatoes. , 2006, Journal of plant physiology.

[35]  A. Barta,et al.  Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins , 2006, Nucleic acids research.

[36]  Hank C Wu,et al.  Experimental validation of novel genes predicted in the un-annotated regions of the Arabidopsis genome , 2007, BMC Genomics.

[37]  V. Walbot,et al.  Nuclear pre-mRNA processing in higher plants. , 1994, Progress in nucleic acid research and molecular biology.

[38]  Andrea Barta,et al.  Genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana. , 2002, Nucleic acids research.

[39]  Howard M. Goodman,et al.  The Arabidopsis splicing factor SR1 is regulated by alternative splicing , 2000, Plant Molecular Biology.

[40]  A. Reddy,et al.  Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. , 2007, The Plant journal : for cell and molecular biology.

[41]  Xiang-Dong Fu,et al.  The superfamily of arginine/serine-rich splicing factors. , 1995, RNA.

[42]  K. Tsai,et al.  Wobble Splicing Reveals the Role of the Branch Point Sequence-to-NAGNAG Region in 3′ Tandem Splice Site Selection , 2007, Molecular and Cellular Biology.

[43]  Andrea Barta,et al.  Evolutionary conservation of minor U12-type spliceosome between plants and humans. , 2005, RNA.

[44]  A. Krainer,et al.  atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes. , 1999, Genes & development.

[45]  A. Barta,et al.  A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions? , 2004, Biochemical Society transactions.

[46]  Kei Iida,et al.  Alternative splicing at NAGNAG acceptor sites shares common properties in land plants and mammals. , 2008, Molecular biology and evolution.

[47]  Stephen M. Mount,et al.  Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. , 2003, Nucleic acids research.

[48]  A. Barta,et al.  Characterization of a novel arginine/serine-rich splicing factor in Arabidopsis. , 1996, The Plant cell.

[49]  Jun Kawai,et al.  A Simple Physical Model Predicts Small Exon Length Variations , 2006, PLoS genetics.

[50]  A. Krainer,et al.  Listening to silence and understanding nonsense: exonic mutations that affect splicing , 2002, Nature Reviews Genetics.

[51]  Rolf Backofen,et al.  Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity , 2004, Nature Genetics.