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.
Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USADepartment of Computer Science and Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA.
Julie
J
Thomas
Saiprasad G
SG
Palusa
Kasavajhala V S K
KV
Prasad
Gul Shad
GS
Ali
Giridara-Kumar
GK
Surabhi
Asa
A
Ben-Hur
Salah E
SE
Abdel-Ghany
Anireddy S N
AS
Reddy
D017398:Alternative Splicing; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics*; D001120:Arginine; D001483:Base Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D006706:Homeostasis; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011523:Protoplasts; D012322:RNA Precursors / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine
D029681:Arabidopsis Proteins; D012322:RNA Precursors; D018749:RNA, Plant; D011994:Recombinant Proteins; D012694:Serine; D001120:Arginine