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Identified of a novel cis -element regulating the alternative splicing of LcDREB2

View Article: PubMed Central - PubMed

ABSTRACT

Alternative splicing (AS) is an important gene regulation mechanism in plants. Despite the widespread use of AS in plant gene expression regulation, the identification of the cis-elements involved in the AS mechanism is rarely reported in plants. To explore the regulation mechanism of the AS of LcDREB2, a DREB2 ortholog from Sheepgrass (Leymus chinensis), the genomic sequences of LcDREB2 and its homologs in Poaceae were aligned, and six mutations were introduced in the conserved sequence of LcDREB2. By analyzing the distinct transcript patterns of the LcDREB2 mutants in transgenic Oryza sativa, a novel cis-element that affected the AS of LcDREB2 was identified as Exonic Splicing Enhancer 1 (ESE1). In addition, five serine-arginine rich (SR) proteins were confirmed to interact with ESE1 by electrophoretic mobility shift assay (EMSA). To further explore the expression regulation mechanism of the DREB subfamily, phylogenetic analysis of DREB2 paralogous genes was performed. The results strongly supported the hypothesis that AS is conserved in Poaceae plants and that it is an evolutionary strategy for the regulation of the functional expression of genes. The findings and methods of our study will promote a substantial step forward in understanding of the plant AS regulation mechanism.

No MeSH data available.


Secondary structure of wild-type and mutated exon 3.(a) The secondary structure of wild-type exon 3 for comparison. (b) The secondary structures of the M4, M5 and M6 mutations of exon 3: no apparent changes in secondary structure were observed. (c) The secondary structures of the M1, M2 and M3 mutations of exon 3: significant changes were observed in these secondary structures.
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f5: Secondary structure of wild-type and mutated exon 3.(a) The secondary structure of wild-type exon 3 for comparison. (b) The secondary structures of the M4, M5 and M6 mutations of exon 3: no apparent changes in secondary structure were observed. (c) The secondary structures of the M1, M2 and M3 mutations of exon 3: significant changes were observed in these secondary structures.

Mentions: To identify whether the primary sequence or the secondary structure of the cis-element on exon 3 was involved in AS regulation, we analyzed the secondary structure of exon 3 and its mutated sequences by energy minimization modeling using the program Sfold. The exon 3 of wild-type LcDREB2 had several pseudoknots in the middle sequences of exon 3, and the other sequences formed RNA-RNA pairs. The integral structure was a long strip, and there was a small knot-like protuberance. Both ends of exon 3 were located together at one end of the structure (Fig. 5a). Meanwhile, the secondary structure of exon 3 in M4, M5 and M6 mutants had no obvious changes compared with the wild type (Fig. 5b). They all formed a long strip with several small pseudoknots. However, the secondary structure of exon 3 in M1, M2 and M3 mutants did show obviously changes (Fig. 5c). M1 contained a large pseudoknot, and the integral structure presented a “Y” shape. M2 had more pseudoknots and two small knot-like protuberances, which changed the integral structure. M3 had a large and obvious pseudoknot compared with the wild type.


Identified of a novel cis -element regulating the alternative splicing of LcDREB2
Secondary structure of wild-type and mutated exon 3.(a) The secondary structure of wild-type exon 3 for comparison. (b) The secondary structures of the M4, M5 and M6 mutations of exon 3: no apparent changes in secondary structure were observed. (c) The secondary structures of the M1, M2 and M3 mutations of exon 3: significant changes were observed in these secondary structures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382683&req=5

f5: Secondary structure of wild-type and mutated exon 3.(a) The secondary structure of wild-type exon 3 for comparison. (b) The secondary structures of the M4, M5 and M6 mutations of exon 3: no apparent changes in secondary structure were observed. (c) The secondary structures of the M1, M2 and M3 mutations of exon 3: significant changes were observed in these secondary structures.
Mentions: To identify whether the primary sequence or the secondary structure of the cis-element on exon 3 was involved in AS regulation, we analyzed the secondary structure of exon 3 and its mutated sequences by energy minimization modeling using the program Sfold. The exon 3 of wild-type LcDREB2 had several pseudoknots in the middle sequences of exon 3, and the other sequences formed RNA-RNA pairs. The integral structure was a long strip, and there was a small knot-like protuberance. Both ends of exon 3 were located together at one end of the structure (Fig. 5a). Meanwhile, the secondary structure of exon 3 in M4, M5 and M6 mutants had no obvious changes compared with the wild type (Fig. 5b). They all formed a long strip with several small pseudoknots. However, the secondary structure of exon 3 in M1, M2 and M3 mutants did show obviously changes (Fig. 5c). M1 contained a large pseudoknot, and the integral structure presented a “Y” shape. M2 had more pseudoknots and two small knot-like protuberances, which changed the integral structure. M3 had a large and obvious pseudoknot compared with the wild type.

View Article: PubMed Central - PubMed

ABSTRACT

Alternative splicing (AS) is an important gene regulation mechanism in plants. Despite the widespread use of AS in plant gene expression regulation, the identification of the cis-elements involved in the AS mechanism is rarely reported in plants. To explore the regulation mechanism of the AS of LcDREB2, a DREB2 ortholog from Sheepgrass (Leymus chinensis), the genomic sequences of LcDREB2 and its homologs in Poaceae were aligned, and six mutations were introduced in the conserved sequence of LcDREB2. By analyzing the distinct transcript patterns of the LcDREB2 mutants in transgenic Oryza sativa, a novel cis-element that affected the AS of LcDREB2 was identified as Exonic Splicing Enhancer 1 (ESE1). In addition, five serine-arginine rich (SR) proteins were confirmed to interact with ESE1 by electrophoretic mobility shift assay (EMSA). To further explore the expression regulation mechanism of the DREB subfamily, phylogenetic analysis of DREB2 paralogous genes was performed. The results strongly supported the hypothesis that AS is conserved in Poaceae plants and that it is an evolutionary strategy for the regulation of the functional expression of genes. The findings and methods of our study will promote a substantial step forward in understanding of the plant AS regulation mechanism.

No MeSH data available.