Role of Polyadenylation in Nucleocytoplasmic Transport of mRNA

作者：Yingqun Huang, Gordon Carmichael · 发表于：Molecular and Cellular Biology · 年份：1996 · DOI：10.1128/mcb.16.4.1534 · 被引用次数：230 · 研究领域：RNA Research and Splicing、HIV Research and Treatment、RNA modifications and cancer

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a lon...