Adema G J, van Hulst K L, Baas P D
Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, The Netherlands.
Nucleic Acids Res. 1990 Sep 25;18(18):5365-73. doi: 10.1093/nar/18.18.5365.
The human calcitonin/CGRP-I (CALC-I) gene contains 6 exons and encodes two polypeptide precursors. In thyroid C-cells, calcitonin (CT) mRNA is produced by splicing of exons 1-2-3 to exon 4 (CT-encoding) and polyadenylation at exon 4. CGRP-I mRNA is produced in particular neural cells by splicing of exons 1-2-3 to exon 5 (CGRP-I-encoding) and the polyadenylated exon 6. We previously reported that model precursor RNAs containing the exon 3 to exon 5 region of the CALC-I gene are processed predominantly into CGRP-I mRNA in vitro, in nuclear extracts of several cell types (neural and non-neural). Using truncated precursor RNAs containing only the exon 3 to exon 4 region of the CALC-I gene it was shown that CT splicing is an inefficient reaction in which a uridine residue serves as the major site of lariat formation. Here we report that the low CT splicing efficiency and the dominance of CGRP-I splicing over CT splicing in vitro are primarily due to the usage of the CT-specific uridine branch acceptor. Mutation of this uridine residue into an adenosine residue resulted in a strong increase in CT splicing efficiency causing a reversal of the splicing pattern. In addition, it was shown that this point mutation also increased CT splicing efficiency in vivo. These results and data obtained from other experiments involving mutation of the CT splice acceptor site suggest that the uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of the CALC-I pre-mRNA.
人降钙素/CGRP-I（CALC-I）基因包含6个外显子，编码两种多肽前体。在甲状腺C细胞中，降钙素（CT）mRNA是通过外显子1-2-3与外显子4（编码CT）拼接，并在外显子4处进行聚腺苷酸化产生的。CGRP-I mRNA是在特定神经细胞中通过外显子1-2-3与外显子5（编码CGRP-I）拼接以及聚腺苷酸化的外显子6产生的。我们之前报道过，在几种细胞类型（神经细胞和非神经细胞）的核提取物中，包含CALC-I基因外显子3至外显子5区域的模型前体RNA在体外主要加工成CGRP-I mRNA。使用仅包含CALC-I基因外显子3至外显子4区域的截短前体RNA表明，CT拼接是一个低效反应，其中尿苷残基是套索形成的主要位点。在此我们报道，体外CT拼接效率低以及CGRP-I拼接在CT拼接中占主导地位主要是由于CT特异性尿苷分支受体的使用。将该尿苷残基突变为腺苷残基导致CT拼接效率大幅提高，从而使拼接模式发生逆转。此外，还表明该点突变在体内也提高了CT拼接效率。这些结果以及从其他涉及CT剪接受体位点突变的实验中获得的数据表明，尿苷分支受体是一个参与CALC-I前体mRNA可变加工调控的顺式作用元件。
