Polypyrimidine tract‐binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control

Alternative splicing (AS) of precursor mRNAs is a widespread phenomenon in plants; however, many questions, especially regarding its regulation and functional implications, remain to be elucidated. In vertebrates, polypyrimidine tract‐binding proteins (PTBs) have been identified as key splicing factors influencing splice site selection and orchestrating coordinated splicing programmes during developmental processes. Here, we analysed three PTB homologues from Arabidopsis thaliana and provide evidence for their gene regulatory potential based on AS and a splicing‐independent mechanism. Our data reveal that Arabidopsis PTB homologues are subject to extensive auto‐ and cross‐regulation via AS‐coupled nonsense‐mediated decay, thereby establishing a basis for interlinking their expression. Furthermore, the multiple modes of action of Arabidopsis PTB homologues are reflected in their subcellular localization in the nucleus, cytosol and processing bodies. This work provides insight into the regulation of AS in plants and highlights the regulatory potential of the multifunctional plant PTB homologues, which might have important implications in diverse biological processes.     

Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor Requires a Suboptimal Upstream 3′ Splice Site

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3′ splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3′ splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5′ end, a C-rich central part, and an AG-rich 3′ end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691–4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3′ splice site in the BPV-1 late pre-mRNA, Rous sarcoma virus src pre-mRNA, human immunodeficiency virus tat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3′ splice site, and in the human β-globin pre-mRNA, which contains a constitutive 3′ splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3′ splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.     

Arabidopsis ROOT INITIATION DEFECTIVE1, a DEAH-Box RNA Helicase Involved in Pre-mRNA Splicing,Is Essential for Plant Development

Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. A multitude of proteins are known to be involved in pre-mRNA splicing in plants; however, the physiological roles of only some of these have been examined. Here, we investigated the developmental roles of a pre-mRNA splicing factor by analyzing root initiation defective1-1 (rid1-1), an Arabidopsis thaliana mutant previously shown to have severe defects in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high-temperature conditions. Phenotypic analysis in planta indicated that RID1 is differentially required during development and has roles in processes such as meristem maintenance, leaf morphogenesis, and root morphogenesis. RID1 was identified as encoding a DEAH-box RNA helicase implicated in pre-mRNA splicing. Transient expression analysis using intron-containing reporter genes showed that pre-mRNA splicing efficiency was affected by the rid1 mutation, which supported the presumed function of RID1 in pre-mRNA splicing. Our results collectively suggest that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development.     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Regulation of glutamate receptor B pre-mRNA splicing by RNA editing

RNA-editing enzymes of the ADAR family convert adenosines to inosines in double-stranded RNA substrates. Frequently, editing sites are defined by base-pairing of the editing site with a complementary intronic region. The glutamate receptor subunit B (GluR-B) pre-mRNA harbors two such exonic editing sites termed Q/R and R/G. Data from ADAR knockout mice and in vitro editing assays suggest an intimate connection between editing and splicing of GluR-B pre-mRNA.

By comparing the events at the Q/R and R/G sites, we can show that editing can both stimulate and repress splicing efficiency. The edited nucleotide, but not ADAR binding itself, is sufficient to exert this effect. The presence of an edited nucleotide at the R/G site reduces splicing efficiency of the adjacent intron facilitating alternative splicing events occurring downstream of the R/G site.

Lack of editing inhibits splicing at the Q/R site. Editing of both the Q/R nucleotide and an intronic editing hotspot are required to allow efficient splicing. Inefficient intron removal may ensure that only properly edited mRNAs become spliced and exported to the cytoplasm.

     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Molecular and cytological characterization of ZTL in <Emphasis Type="Italic">Ipomoea nil</Emphasis>

The ZEITLUPE (ZTL) protein is involved in the control of circadian period, hypocotyl elongation and flowering time in Arabidopsis thaliana. The aim of the present work was the identification of the InZTL gene and localization of its mRNA in the model short-day plant Ipomoea nil. The deduced InZTL protein of 622 amino acid residues contained a LOV domain at the N-terminal part, followed by an F-box domain and six carboxy terminal kelch repeats. Amino acid sequence of InZTL showed 84 % homology with Mesembryanthemum crystallinum ZTL (McZTL) and 83 % with Arabidopsis thaliana ZTL (AtZTL). Fluorescence in situ hybridization (FISH) to InZTL mRNA showed its high accumulation in the vascular bundles as well in the guard cells of the cotyledon. Immunolocalization of ZTL protein indicated a similar distribution pattern of ZTL protein as InZTL mRNAs.     

Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.     

Genome-wide identification of NBS-encoding resistance genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.     

Evolutionary Process of the Genomic Sequence Around the 100 Map Unit of Chromosome 1 in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Comparative analysis of nucleotide sequences of the genomic region located around 100 map unit of chromosome 1 using two accessions, Columbia (Col) and Landsberg erecta (Ler), of Arabidopsis thaliana was performed. High divergence was detected between them, and the length of the Ler sequence was half of corresponding sequence of Col. This divergence occurred by tandem duplication, deletion of large regions, and insertion of unrelated sequences. These events led to the high polymorphism of plant disease resistant genes, which are located in the analyzed region. It is highly probable that two-round duplication occurred, and the insertion sequences are transposable elements. The data suggest that the analyzed region had been evolving until quite recently.     

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1–1 was a G→A change 4 nt upstream from the 3′ splice site of intron 5, while the mutation in cop1–2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5′ splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1–8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3′ splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.     

Genomic neighborhoods for <Emphasis Type="Italic">Arabidopsis</Emphasis>retrotransposons: a role for targeted integration in the distribution of the Metaviridae

Background  

Retrotransposons are an abundant component of eukaryotic genomes. The high quality of the Arabidopsis thaliana genome sequence makes it possible to comprehensively characterize retroelement populations and explore factors that contribute to their genomic distribution.     

Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.