Svensson C, Pettersson U, Akusjärvi G
J Mol Biol. 1983 Apr 15;165(3):475-95. doi: 10.1016/s0022-2836(83)80214-9.
The r-strand of early region 1A (E1A) of adenovirus serotype 2 is transcribed into three completely overlapping messenger RNA species, a 9S, a 12S and a 13S mRNA. These three mRNAs are processed from a common colinear RNA precursor and differ only with regard to the size of the intron removed during mRNA maturation. We have studied the processing pathways for the E1A mRNAs by using an assay for transient expression of recombinant plasmids containing the E1A region. All three region E1A mRNAs are synthesized and transported to the cytoplasm in sufficient quantities to permit a detailed study of their structure by S1 endonuclease analysis and primer extension. Additionally, we show that the 72 base-pair repeat from simian virus 40 (SV40), when located upstream of the E1A promoter stimulates expression of the E1A mRNAs five- to tenfold. In order to determine whether splicing of the E1A mRNAs is sequential, i.e. whether the 13S and 12S mRNAs can serve as intermediates in splicing, we constructed two plasmids that lack the intervening sequences that are removed during the maturation of the 12S and 13S mRNAs, respectively. From an analysis of the RNAs produced after transfection with these deletion mutants, the following major conclusions can be made. (1) Splicing of the E1A mRNAs is non-sequential, i.e. the 13S, 12S and 9S RNAs are generated by separate splicing events using the nuclear colinear transcript as the only precursor RNA. (2) RNA splicing is not a prerequisite for an efficient transport of the E1A mRNAs to the cytoplasm. (3) The 13S RNA can be further processed to 12S and 9S RNA species. These splicing events are, however, illegitimate and give rise to 12S and 9S RNAs that both lack one nucleotide at the splice junction. (4) A coupling between splicing and nuclear transport is most likely required in vivo to prevent illegitimate splicing of the 13S mRNA. (5) The 12S RNA does not serve as a precursor for further processing to the 9S RNA. (6) Splicing of the E1A mRNAs followed strictly the G-T-A-G rule.
腺病毒2型早期区域1A（E1A）的r链转录成三种完全重叠的信使RNA种类，即9S、12S和13S mRNA。这三种mRNA由一个共同的共线性RNA前体加工而来，仅在mRNA成熟过程中去除的内含子大小方面有所不同。我们通过对含有E1A区域的重组质粒进行瞬时表达分析，研究了E1A mRNA的加工途径。所有三种区域E1A mRNA都能合成并大量转运到细胞质中，从而可以通过S1核酸酶分析和引物延伸对其结构进行详细研究。此外，我们还表明，来自猴病毒40（SV40）的72个碱基对重复序列，当位于E1A启动子上游时，可将E1A mRNA的表达刺激5至10倍。为了确定E1A mRNA的剪接是否是连续的，即13S和12S mRNA是否可作为剪接中间体，我们构建了两个质粒，分别缺失在12S和13S mRNA成熟过程中去除的间隔序列。通过对这些缺失突变体转染后产生的RNA进行分析，可得出以下主要结论。（1）E1A mRNA的剪接不是连续的，即13S、12S和9S RNA是通过单独的剪接事件产生的，以核共线性转录本作为唯一的前体RNA。（2）RNA剪接不是E1A mRNA有效转运到细胞质的先决条件。（3）13S RNA可进一步加工成12S和9S RNA种类。然而，这些剪接事件是非法的，产生的12S和9S RNA在剪接连接处都缺少一个核苷酸。（4）体内很可能需要剪接与核转运之间的偶联，以防止13S mRNA的非法剪接。（5）12S RNA不作为进一步加工成9S RNA的前体。（6）E1A mRNA的剪接严格遵循G-T-A-G规则。
