Alonso-Caplen F V, Krug R M
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Mol Cell Biol. 1991 Feb;11(2):1092-8. doi: 10.1128/mcb.11.2.1092-1098.1991.
Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.
流感病毒NS1 mRNA由宿主核酶进行剪接以形成NS2 mRNA，并且这种剪接在受感染细胞中受到调控，使得剪接后的NS2 mRNA的稳态量仅为未剪接的NS1 mRNA的稳态量的约10%。预计这种调控是由于剪接速率的抑制以及未剪接的NS1 mRNA从细胞核的有效转运所致。为了确定NS1 mRNA的剪接速率是否受流感病毒感染细胞中的反式因子控制，将NS1基因插入腺病毒载体中。用[3H]尿苷脉冲标记法测量了感染该载体的细胞和流感病毒感染细胞中NS1 mRNA的剪接速率。两个系统中NS1 mRNA的剪接速率没有显著差异，强烈表明流感病毒感染细胞中NS1 mRNA的剪接速率仅由NS1 mRNA自身的顺式作用序列控制。与剪接速率相反，腺病毒重组体感染的细胞中NS1 mRNA的剪接程度相对于流感病毒感染细胞中发生的剪接程度显著增加。这如果不是完全归因于重组体感染细胞中未剪接的NS1 mRNA的核质转运受阻，也可在很大程度上归因于此。大多数未剪接的NS1 mRNA存在于细胞核部分，并且没有检测到NS1蛋白的合成。当NS1 mRNA的3'剪接位点通过突变失活时，NS1 mRNA被转运并翻译，表明转运受阻是因为NS1 rRNA进入了剪接途径。这种转运受阻在流感病毒感染细胞中显然被消除了。这些实验证明了未剪接的NS1 mRNA的核质转运在调节NS1 mRNA的剪接程度中的重要作用。
