A novel mechanism of aberrant pre-mRNA splicing in humans.
A novel mechanism of aberrant pre-mRNA splicing in humans.
复制标题
人类异常前 mRNA 剪接的新机制。
DOI：
10.1093/hmg/6.6.909
复制
发表时间：
1997
影响因子：
3.5
通讯作者：
Phillips3rd,JA
中科院分区：
文献类型：
--
作者：
Cogan,JD;Prince,MA;Lekhakula,S;Bundey,S;Futrakul,A;McCarthy,EM;Phillips3rd,JA
真核生物前体mRNA剪接受内含子边界和分支位点的共有序列调控。最近，Sirand-Pugnetet等人报道了一个额外的内含子序列的重要性，即鸡β-原肌球蛋白中的（A/U）GGG重复序列，它是剪接体组装所需蛋白质的结合位点。有趣的是，我们已经检测到人类生长激素（GH）基因的IVS 3突变，这些突变影响了一个假定的同源共有序列，并且也干扰了剪接。在一系列引起外显子跳跃的显性负性GH突变中，我们发现了两个不在5′和3′剪接位点或分支共有位点内发生的突变。第一个突变是人GH基因IVS 3第28位碱基G→A（+28 G → A）的转换，第二个突变是IVS 3第18位碱基缺失（del+28-45）。这些突变分离与常染色体显性GH缺乏症在两个kinetics和没有其他等位基因GH基因的变化被检测到。来自含有+28G的A2 A或del+28-45等位基因的表达载体的转录物的RT-PCR扩增产生的产物显示选择性剪接的>10倍优选使用，类似于先前报道的IVS 3供体位点突变的发现。这两个突变都位于5′剪接位点下游28 bp处，对干扰序列的检查显示，内含子XGGG重复序列与在鸡β-原肌球蛋白中发现的调节mRNA剪接的重复序列相似。有趣的是，我们的突变中涉及的XGGG重复序列与所谓的“赢家”RNA序列中的重复序列具有同源间隔。前体mRNA转录物中的“赢家”序列与A1异质核核糖核蛋白（hnRNP）的结合被认为在前体mRNA包装和运输以及包含多个5 '剪接位点的前体mRNA的5'剪接位点选择中发挥重要作用。我们的研究结果表明：（i）XGGG重复序列可能调节人GH基因的选择性剪接，（ii）这些重复序列的突变通过干扰选择性剪接导致GH缺陷。同源内含子序列的突变可能是其他人类疾病的基础。
Eukaryotic pre-mRNA splicing is regulated by consensus sequences at the intron boundaries and branch site. Recently, Sirand-Pugnetet al.reported the importance of an additional intronic sequence, an (A/U)GGG repeat in chicken β-tropomyosin that is a binding site for a protein required for spliceosome assembly. Interestingly, we have detected mutations in IVS3 of the human growth hormone (GH) gene that affect a putative, homologous consensus sequence and which also perturb splicing. In a series of dominant-negative GH mutations that cause exon skipping, we found two mutations that do not occur within the 5′ and 3′ splice sites, or branch consensus sites. The first mutation is a G→A transition of the 28th base (+28G→A) of and the second deletes 18 bp (del+28–45) of IVS3 of the human GH gene. These mutations segregated with autosomal dominant GH deficiency in both kindreds and no other allelic GH gene changes were detected. RT-PCR amplification of transcripts from expression vectors containing the +28G→A or del+28–45 alleles yielded products showing a >10-fold preferred use of alternative splicing, similar to findings previously reported for IVS3 donor site mutations. Both mutations are located 28 bp downstream from the 5′ splice site and examination of the sequences perturbed revealed an intronic XGGG repeat similar to the repeat found to regulate mRNA splicing in chicken β-tropomyosin. Interestingly, the XGGG repeats involved in our mutations exhibit homologous spacing to those in a so-called ‘winner’ RNA sequence. Binding of A1 heterogeneous nuclear ribonucleoprotein (hnRNP) by ‘winner’ sequences in pre-mRNA transcripts is thought to play an important role in pre-mRNA packaging and transport as well as 5′ splice site selection in pre-mRNAs that contain multiple 5′ splice sites. Our findings suggest that (i) XGGG repeats may regulate alternative splicing in the human GH gene and (ii) mutations of these repeats cause GH deficiency by perturbing alternative splicing. Mutations of homologous intron sequences may underlie other human diseases.