The predominant cAMP-stimulated 3.5 kb StAR mRNA contains specific sequence elements in the extended 3'UTR that confer high basal instability
The predominant cAMP-stimulated 3.5 kb StAR mRNA contains specific sequence elements in the extended 3'UTR that confer high basal instability
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DOI：
10.1677/jme.1.02153
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发表时间：
2007-02-01
影响因子：
3.5
通讯作者：
Jefcoate, Colin R.
中科院分区：
文献类型：
--
作者：
Duan, Haichuan;Jefcoate, Colin R.
cAMP刺激啮齿动物类固醇生成细胞产生两种星星转录物，一种主要的3.5 kb和一种次要的1 kb。6 kb mRNA，仅在它们的31个非翻译区（3 'UTR）上不同。它们对8-Br-cAMP的刺激和去除表现出非常不同的反应，3.5 kb形式的增加和下降比1.6 kb形式快得多。3（.）5 kb星星mRNA包含三个保守的富含AU的元件（AURE）基序，介导人类基因组中超过900个基因的快速mRNA周转。在本文中，我们探讨了转录后调控类固醇和nonsteroidogenic细胞使用表达载体含有星星或荧光素酶与不同的星星3 'UTR。我们发现，星星或荧光素酶蛋白和mRNA的基础稳态水平在3 'UTR为3（.）5 kb的星星与1（.）6kb 3'UTR。通过直接mRNA转染检测转录物稳定性显示3（.）5 kb mRNA相对于1（.）6 kb mRNA。然而，尽管从相同的启动子转录，长的3 'UTR导致成熟细胞质mRNA的出现率降低了5倍。这是由于在输出到细胞质之前，不成熟转录物的核加工效率较低。选择性3 'UTR序列取代、缺失和突变表明，这种表达缺失是由700个碱基的基础不稳定区中的特异性序列和非特异性长度效应叠加产生的。这些机制在类固醇生成细胞中被选择性地增强。AURE对类固醇生成细胞的选择性基础去稳定作用较小，其通过突变而被去除。在3.5 kb星星载体中包含内含子增强星星表达，表明内含子复合物对核加工的影响。Br-cAMP通过减弱延伸的3 'UTR内的细胞核和细胞质不稳定性机制，提供了不依赖于转录而快速调节星星表达的额外手段。
cAMP stimulation of rodent steroidogenic cells produces two StAR transcripts, a major 3.5 kb and a minor 1(.)6 kb mRNA, differing only in their 31 untranslated regions (3'UTR). They exhibit very different responses to stimulation and removal of 8-Br-cAMP, with the 3.5 kb form increasing and declining much more rapidly than the 1.6 kb form. The 3' end of the 3(.)5 kb StAR mRNA contains three conserved AU-rich element (AURE) motifs that mediate fast mRNA turnover in over 900 genes in the human genome. In this paper, we explore post-transcriptional regulation in steroidogenic and nonsteroidogenic cells using expression vectors containing StAR or luciferase with different StAR 3'UTRs. We show that the basal steady-state levels of StAR or luciferase protein and mRNA are five to eight times lower with the 3'UTR of 3(.)5 kb StAR compared with that of the 1(.)6 kb 3'UTR. Examination of transcript stability by direct mRNA transfection showed only a 1.5-fold increase in the rate of cytoplasmic decay of the 3(.)5 kb mRNA relative to the 1(.)6 kb mRNA. However, the long 3'UTR caused a fivefold decrease in the rate of appearance of mature cytoplasmic mRNA despite transcription from the same promoter. This is attributed to less efficient nuclear processing of immature transcripts prior to export to cytoplasm. Selective 3'UTR sequence substitutions, deletions, and mutations showed that this loss of expression is produced additively by specific sequences in a 700-base basal instability region and by non-specific length effects. These mechanisms are selectively enhanced in steroidogenic cells. The AURE contribute a smaller basal destabilization effect selective for steroidogenic cells that is removed by their mutations. Inclusion of introns in the 3.5 kb StAR vector enhances StAR expression, suggesting the effects of introns complexes on nuclear processing. Br-cAMP provides an additional means to rapidly modulate StAR expression independent of transcription by attenuating the nuclear and cytoplasmic instability mechanisms within the extended 3'UTR.