Functional analysis of three Arabidopsis SR proteins (SCl33, SC35, SCl30a) in plant development and splicing

Precursor-mRNA (Pre-mRNA) splicing is dependent on many RNA binding proteins that recognize sequence signals in RNA and regulate splicing. The serine/arginine (SR)-rich proteins are a family of RNA binding spliceosomal proteins that perform essential functions during spliceosome assembly by interacting with splicing regulatory sequences in pre-mRNA as well as with other spliceosomal proteins. These RNA-protein and protein-protein interactions of SRs play a crucial role in constitutive splicing as well as in alternative splicing (AS). Since SR genes regulate their own splicing and subsequently affect AS of other SR mRNAs as well as that of many other coding genes, elucidation of functions of the SRs is critical for understanding gene regulation at the pre-mRNA splicing level.;To address the role of SRs in plant development we generated loss-of-function mutants of SC35, the sole member of the SC35 family with a counterpart in humans, and of SCl33 and SCl30a that belong to the plant-specific SC35-like (SCL) family. To address potential functional redundancy and/or synthetic phenotypes among these genes we generated three double mutants (sc35 scl30a, sc35 scl33, scl33 scl30a) representing all combinations, and a triple mutant. All mutants are viable but displayed complex and opposing flowering phenotypes in different mutants. Among the single mutants sc35 and scl30a showed early flowering whereas scl33 showed delayed flowering under both long days (LD) and short days (SD). In double mutant combinations, sc35 scl30a flowered early as in single mutants and no additive effect was observed. In contrast scl33 was epistatic to scl30a in the double mutant and to sc35 and scl30a in the triple mutant and these exhibited an even more pronounced late flowering phenotype as compared to scl33. The late flowering phenotype of scl33, scl33 scl30a and scl33 sc35 scl30a under both LD and SD and rescue of this phenotype by vernalization, suggest that they regulate the autonomous flowering pathway. In the late flowering mutants expression of Flowering Locus C (FLC), a key negative regulator of flowering, and FRIGIDA (FRI), a positive regulator of FLC expression are upregulated. In contrast, early flowering mutants (sc35, scl30a and sc35 scl30a) showed increased expression of FLOWERING LOCUS T (FT), a positive regulator of flowering. These results indicate that members of the SR gene family perform opposing roles in regulating flowering time.;In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing. However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. To study the role of SR proteins in AS, a splicing reporter (GFP--intron--GFP) was constructed, 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 its 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. Thus, 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. (Abstract shortened by UMI.).