Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, USA.
BMC Mol Biol. 2009 Nov 17;10:103. doi: 10.1186/1471-2199-10-103.
The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid.
A candidate cis-acting exonic splicing silencer (ESS) comprising nucleotides 22-30 of exon 6A sequence was identified corresponding to the a silencer consensus sequence of AAGGGG. The function of this sequence as an ESS was confirmed in vivo both in the context of the reporter minigene as a plasmid and in the context of a longer minigene with VEGF exon 6A in its native context in an adenoviral gene transfer vector. Further mutagenesis studies resulted in the identification of the second G residue of the putative ESS as the most critical for function.
This work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.
血管内皮生长因子（VEGF）的不同同工型在血管生长、结构和功能中发挥不同的作用。VEGF 基因的选择性剪接导致三种丰富同工型的表达：VEGF121、VEGF165 和 VEGF189。VEGF189 的 mRNA 包含选择性剪接的外显子 6A，而 VEGF165 的 mRNA 则缺乏该外显子。本研究的目的是鉴定控制外显子 6A 利用的顺式元件。构建了一个报告基因 minigene（pGFP-E6A），其中包含 GFP 的编码序列，其翻译依赖于 VEGF 外显子 6A 的准确剪接去除。为了鉴定顺式作用的剪接元件，在 pGFP-E6A 质粒中外显子 6A 上进行了顺序缺失。
鉴定出一个候选顺式作用的外显子剪接沉默子（ESS），包含外显子 6A 序列的 22-30 个核苷酸，对应于 AAGGGG 的沉默子保守序列。该序列作为 ESS 的功能在体内得到了证实，无论是在报告基因 minigene 作为质粒的情况下，还是在更长的 minigene 中，VEGF 外显子 6A 在其天然腺病毒基因转移载体中的情况下。进一步的突变研究确定了假定 ESS 中的第二个 G 残基是功能最关键的。
这项工作确定了调节 VEGF 选择性剪接的顺式序列的身份，并决定了 VEGF 同工型的相对表达水平。
