Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.
Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.
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外显子 2 和 3 中的编码元件针对肌原性分化过程中 c-myc mRNA 的下调。
DOI：
10.1128/mcb.17.5.2698
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发表时间：
1997
影响因子：
5.3
通讯作者：
Lee,WM
中科院分区：
文献类型：
--
作者：
Yeilding,NM;Lee,WM
c-mycproto-癌基因表达下调是小鼠C2C12成肌细胞向多核肌管分化的早期分子事件。在分化过程中，尽管c-mycmRNA的转录率没有变化，但其水平下降了3- 10倍。为了鉴定在肌发生过程中靶向c-mycmRNA下调的顺式作用元件，我们用突变mycgenes或嵌合基因稳定转染C2C12细胞，其中各种mycgenes序列与人β-珠蛋白基因或细菌氯霉素乙酰转移酶（CAT）基因融合。在mycmrna的肌原分化过程中，删除mycmrna 2或3外显子的编码序列可以消除mycmrna的下调，而删除5 ‘或3 ’非翻译区（UTRs）的内含子或序列则不会，这表明2和3外显子的编码元件都是mycmrna下调所必需的。将myc3 ' UTR序列或来自CAT或核糖体蛋白L32的编码序列与β-珠蛋白mRNA融合，可以下调嵌合mRNA的表达，这表明myc3 ' UTR序列或来自CAT或核糖体蛋白L32的编码序列的融合不会下调嵌合mRNA的表达，这表明myc3 ' UTR序列或核糖体蛋白L32的编码元件特异性下调。这些结果提出了一个明显的悖论，即mycexon 2或mycexon 3中的编码元件足以下调β-珠蛋白mRNA，但单独的元件都不足以下调β-珠蛋白mRNA，这表明β-珠蛋白mRNA的某些特征可能增强了mycexon 2和3的调控特性。并不是所有mRNA都具有类似的调控功能，因为无论是mycexon 2还是mycexon 3与CAT mRNA的融合都不会下调嵌合mRNA的表达，但这两个元素的融合却会下调。我们从这些结果中得出结论，ycmrna下调需要两个调控元件，一个外显子2和一个外显子3。最后，通过一种高灵敏度和特异性的基于pcr的比较mRNA水平的方法，我们证明了由菌丝子2和3介导的下调导致细胞质mRNA水平下降，而不是核mRNA水平下降，这表明调控是核后事件。
Downregulation in expression of the c-mycproto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-mycmRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-mycmRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutantmycgenes or chimeric genes in which variousmycsequences were fused to the human β-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences frommycexon 2 or exon 3 abolished downregulation ofmycmRNA during myogenic differentiation, while deletion of introns or sequences in the 5′ or 3′ untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary formycmRNA downregulation. Fusion of coding sequences from eithermycexon 2 or 3 to β-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion ofmyc3′ UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements inmycexons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in eithermycexon 2 ormycexon 3 are sufficient to confer downregulation onto β-globin mRNA, but neither element alone was sufficient formycmRNA downregulation, suggesting that some feature of β-globin mRNA may potentiate the regulatory properties ofmycexons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of eithermycexon 2 ormycexon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that twomycregulatory elements, one exon 2 and one in exon 3, are required formycmRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated bymycexons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.