Identification of sequences in c-myc mRNA that regulate its steady-state levels.
Identification of sequences in c-myc mRNA that regulate its steady-state levels.
复制标题
鉴定 c-myc mRNA 中调节其稳态水平的序列。
DOI：
10.1128/mcb.16.7.3511
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发表时间：
1996
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;Rehman,MT;Lee,WM
细胞霉素原癌基因的表达水平响应于环境信号而迅速调节，并影响细胞增殖和分化。调控依赖于c-mycmRNA的快速周转，这使得细胞能够快速改变c-mycmRNA水平。为了鉴定导致mycmRNA不稳定性的元件，已经使用了多种方法，所有这些方法都需要干扰正常细胞代谢的操作。这些不同的方法涉及mRNA的不同区域，并导致缺乏共识，哪些区域实际上决定了快速周转和低稳态水平的c-mycmRNA。为了通过一种不严重干扰细胞代谢并直接评估c-mycmRNA区域对c-mycmRNA稳态水平的影响的方法来识别这些区域，我们开发了一种使用逆转录和PCR的检测方法，以比较从两个类似构建的mycgenes瞬时共转染到增殖的C2 C12成肌细胞中转录的humanmycmRNA的稳态水平。将缺失突变引入到mycgenes中，并将其mRNA水平与接近正常的参考mycmRNA水平进行比较。myc 3 ′非翻译区（UTR）的大部分缺失可提高mycmRNA水平，而myc 5 ′非翻译区（外显子1的大部分）、外显子2或外显子3的蛋白编码区的序列缺失则不能提高mycmRNA水平，这表明3′非翻译区是保持mycmRNA水平较低的原因。使用类似的逆转录-PCR分析比较两种β-globin-myc融合mRNA的稳态水平，我们发现themyc 3 ′ UTR的融合通过使β-globinmRNA不稳定而降低珠蛋白mRNA水平。令人惊讶的是，融合mycexon 3的蛋白编码区也降低了珠蛋白mRNA的稳态水平。通过研究外显子3编码序列在调节c-mycmRNA转换中可能发挥的其他作用，我们证明了这些序列，而不是myc 3 ′ UTR序列，是成肌细胞分化过程中c-mycmRNA正常转录后下调所必需的。我们的结论是，虽然c-mycmRNA中的两个元件可以作为不稳定的决定因素在异源的情况下，只有在3′ UTR的不稳定元件调节其在增殖C2 C12细胞的稳态水平。
The level of cellularmycproto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-mycmRNA, which enables cells to rapidly alter c-mycmRNA levels. Efforts to identify elements inmycmRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-mycmRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-mycmRNA region on the steady-state levels of c-mycmRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of humanmycmRNAs transcribed from two similarly constructedmycgenes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced intomycgenes, and the levels of their mRNAs were compared with that of a near-normal, referencemycmRNA. Deletion of most of themyc3′ untranslated region (UTR) raisedmycmRNA levels, while deletion of sequences in themyc5′ UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3′ UTR is responsible for keepingmycmRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two β-globin–myc fusion mRNAs, we showed that fusion of themyc3′ UTR lowers globin mRNA levels by destabilizing β-globin mRNA. Surprisingly, fusion of the protein-coding region ofmycexon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-mycmRNA turnover, we demonstrated that these sequences, but notmyc3′ UTR sequences, are necessary for the normal posttranscriptional downregulation of c-mycmRNA during myoblast differentiation. We conclude that, while two elements within c-mycmRNA can act as instability determinants in a heterologous context, only the instability element in the 3′ UTR regulates its steady-state levels in proliferating C2C12 cells.