Multiple transcription initiation sites, alternative splicing, and differential polyadenylation contribute to the complexity of human neurofibromatosis 2 transcripts
Multiple transcription initiation sites, alternative splicing, and differential polyadenylation contribute to the complexity of human neurofibromatosis 2 transcripts
复制标题
DOI：
10.1006/geno.2001.6672
复制
发表时间：
2002-01-01
期刊：
影响因子：
4.4
通讯作者：
Welling, DB
中科院分区：
文献类型：
--
作者：
Chang, LS;Akhmametyeva, EM;Welling, DB
北方印迹分析表明，人神经纤维瘤病2型（NF 2）的cDNA杂交到多个RNA种类。为了检测这些杂交RNA是否代表NF 2转录本，我们通过以下技术的组合克隆了完整的NF 2 cDNA：cDNA末端的5'和3'快速扩增，RT-PCR，以及从IMAGE联盟中搜索和测序NF 2相关cDNA克隆。我们发现，人类NF 2转录起始于多个位置。与先前报道的类似，NF 2转录物在编码外显子中进行选择性剪接。我们分离了八种选择性剪接的NF 2 cDNA亚型，其中一种含有一个新的外显子，称为外显子2 '，它可能编码不同大小的蛋白质。我们组装了重叠的cDNA片段，最长的NF 2 cDNA，包含所有17个外显子，由6067个核苷酸组成，这与与NF 2探针杂交的主要RNA种类的大小一致。该cDNA在ATG起始密码子上游有一个425个核苷酸的5'非翻译区，和一个长3869个核苷酸的3'非翻译区。我们还分离了两个较短的NF 2 cDNA，它们被不同的多聚腺苷酸化信号序列终止，这表明多聚腺苷酸化位点的差异使用也有助于人类NF 2转录物的复杂性。参照转录起始位点的定位，我们分析了人NF 2基因的5'侧翼序列。瞬时转染分析在人293肾，SK-N-AS神经母细胞瘤，和NT 2/D1畸胎瘤细胞与NF 2启动子荧光素酶嵌合体结构揭示了一个核心启动子区延伸400个碱基对的主要转录起始位点。尽管完整的启动子活性需要多个区域，但是定点诱变实验鉴定了富含GC的序列（位置-58至-46），其可以被转录因子Sp1结合，作为正顺式作用调节元件。在果蝇SL 2细胞中的共转染研究表明，Sp1可以通过富含GC的序列激活NF 2启动子。
Northern blot analysis has shown that the human neurofibromatosis type 2 (NF2) cDNA hybridizes to multiple RNA species. To examine whether these hybridizing RNA species represent NF2 transcripts, we cloned the complete NF2 cDNA by a combination of techniques: 5' and 3' rapid amplification of cDNA ends, RT-PCR, and searching and sequencing the NF2-related cDNA clones from the IMAGE consortium. We showed that human NF2 transcripts initiate at multiple positions. Analogous to those reported previously, NF2 transcripts undergo alternative splicing in the coding exons. We isolated eight alternatively spliced NF2 cDNA isoforms, including one that contains a new exon termed exon 2', which potentially could encode proteins of different sizes. We assembled the overlapping cDNA fragments, and the longest NF2 cDNA, containing all 17 exons, consists of 6067 nucleotides, which is consistent with the size of the major RNA species hybridized to the NF2 probe. The cDNA has a 425-nucleotide 5' untranslated region upstream from the ATG start codon, and a long 3' untranslated region of 3869 nucleotides. We also isolated two shorter NF2 cDNAs that were terminated by different polyadenylation signal sequences, which indicates that differential usage of multiple polyadenylation sites also contributes to the complexity of human NF2 transcripts. By reference to the transcription initiation site mapped, we analyzed the 5' flanking sequence of the human NF2 gene. Transient transfection analysis in human 293 kidney, SK-N-AS neuroblastoma, and NT2/D1 teratocarcinoma cells with NF2 promoter-luciferase chimeric constructs revealed a core promoter region extending 400 base pairs from the major transcription initiation site. Although multiple regions are required for full promoter activity, a site-directed mutagenesis experiment identified a GC-rich sequence (position -58 to -46), which could be bound by transcription factor Sp1, as a positive cis-acting regulatory element. Cotransfection studies in Drosophila melanogaster SL2 cells showed that Sp1 could activate the NF2 promoter through the GC-rich sequence.