Environmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay (NMD)-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

Environmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

Identification of an intronic splicing regulatory element involved in auto-regulation of alternative splicing of SCL33 pre-mRNA.

In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.

Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.

Extensive coupling of alternative splicing of pre-mRNAs of serine/arginine (SR) genes with nonsense-mediated decay.

In Arabidopsis, pre-mRNAs encoding serine/arginine (SR) proteins, key regulators of constitutive and alternative splicing, are extensively alternatively spliced. In seedlings, 13 SR genes are alternatively spliced to generate 75 transcripts, of which 53 contain a premature termination codon (PTC). However, it is not known if any of the PTC-containing splice variants are the targets of nonsense-mediated decay (NMD) and if there is any link between NMD and the abundance of functional transcripts. Here, we analyzed the abundances of all splice variants for each alternatively spliced gene in an Arabidopsis mutant that lacks UPF3, one of the core components of NMD machinery, to determine if the PTC-containing transcripts are degraded by NMD. Our results show that about half of the 53 splice variants with a PTC are the targets of degradation by NMD. The accumulation of PTC-containing transcripts resulted in concomitant reduction in the amount of functional transcript. These results show widespread coupling of alternative splicing with NMD in the SR gene family, suggesting a strong link between unproductive splicing and the abundance of functional transcripts.

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

BACKGROUND: Several 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. RESULTS: In 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. CONCLUSION: NAGNAG 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.

Regulation of plant developmental processes by a novel splicing factor.

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.