RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform.
RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform.
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我们先前已经报道，人类ACAT1基因通过对从1号和7号染色体转录的两个不连续RNA进行染色体间加工而产生嵌合mRNA。嵌合mRNA分别以AUG1397-1399和GGC1274-1276为翻译起始密码子，产生正常的50-kD ACAT1和新的具有酶活性的56-kD亚型，该亚型存在于包括人单核细胞来源的巨噬细胞在内的人类细胞中。在这项工作中，我们报告了位于GGC1274-1276密码子附近的RNA二级结构是产生56-kD亚型所必需的。将含有野生型、缺失或突变的茎环序列与部分ACAT1 Aug-Open阅读框(ORF)或其他基因的ORF连锁的表达载体导入细胞，分别检测3个预测的茎环(NT 1255-1268、1286-1342和1355-1384)的效果。用Western印迹分析检测其表达模式。我们发现，5 6kD亚型的产生需要7号染色体的上游茎环1255-1268和1号染色体的下游茎环1286-1342，而1号染色体的最后一个茎环1355-1384是必需的。使用具有稳定发夹的单顺反子和双顺反子载体的实验结果表明，GGC1274-1276密码子的翻译起始是由内部核糖体进入位点(IRES)介导的。进一步的实验表明，GGC1274-1276密码子的翻译起始需要上游具有AU组成的RNA二级结构和下游具有富含GC的二级结构。这一机制工作进一步支持了嵌合人ACAT1基因表达来自两条不同染色体的生物学意义。
We have previously reported that human ACAT1 gene produces a chimeric mRNA through an interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397–1399 and GGC1274–1276 respectively as translation initiation codons to produce the normal 50-kD ACAT1 and a novel enzymatically active 56-kD isoform, which is authentically present in human cells including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC1274–1276 codon are required for producing the 56-kD isoform. The effects of the three predicted stem-loops (nt 1255–1268, 1286–1342 and 1355–1384) were tested individually by transfecting expression plasmids, which contain the wild-type, deleted or mutant stem-loop sequences linked with the partial ACAT1 AUG-open reading frame (ORF) or with the ORFs of other genes, into cells. The expression patterns were monitored by Western blot analyses. We found that the upstream stem-loop1255–1268 from chromosome 7 and downstream stem-loop1286–1342 from chromosome 1 were needed for production of the 56-kD isoform, whereas the last stem-loop1355–1384 from chromosome 1 was dispensable. The results of experiments using both the monocistronic and bicistronic vectors with a stable hairpin showed that the translation initiation from the GGC1274–1276 codon was mediated by internal ribosome entry site (IRES). Further experiments revealed that the translation initiation from the GGC1274–1276 codon required the upstream RNA secondary structure with AU-constitution and the downstream one with GC-richness. This mechanistic work further supports the biological significance that the chimeric human ACAT1 mRNA is expressed from two different chromosomes.
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影响因子：
4.5
作者：
Linnstaedt, Sarah D.;Kasprzak, Wojciech K.;Casey, John L.
通讯作者：
Casey, John L.
DOI：
10.1073/pnas.90.16.7642
发表时间：
1993-08-15
影响因子：
11.1
作者：
HELLEN, CUT;WITHERELL, GW;WIMMER, E
通讯作者：
WIMMER, E
影响因子：
5.3
作者：
Huez, I;Créancier, L;Prats, H
通讯作者：
Prats, H
影响因子：
5.3
作者：
Pestova, TV;Hellen, CUT;Shatsky, IN
通讯作者：
Shatsky, IN
影响因子：
14.9
作者：
Dhar, Debojyoti;Roy, Swagata;Das, Saumitra
通讯作者：
Das, Saumitra