The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180? inverse symmetry, and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region=s known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or sub-region RNAs, and that formation of RNA-protein complexes are conferred by interaction with other novel proteins. UV-crosslinking of RNA-protei n complexes, coupled with RNase T1 digestion and denaturing polyacrylamide gel electrophoresis, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual sub-region RNAs, apparently recognizing the CTE RNA at multiple sites of interaction. In addition, we constructed a series of SRV infectious clone recombinants containing varying permutations of sub-region fragments. RNA slot-blot analyses indicate that recombinants containing the complete CTE in sense orientation, or containing two contiguous sub-regions that reconstitute the 3' two-thirds of this region, can facilitate or partially facilitate SRV RNA export in transfectant cells. These experiments indicate that sequence determinants of the 3' IR, in addition to secondary structure, are important factors in the nuclear export of unspliced SRV RNA, and that proteins other than RNA helicase A may also directly recognize the serogroup 2 SRV CTE. FUNDING NIH DK53462 PUBLICATIONS Li B, Wyman T, Moudgil T, Marracci G, Ju CF, Machida CA. Nuclear export of simian retroviral RNA Critical genetic elements and cellular protein factors. In Program of the Sixteenth Annual Symposium on Nonhuman Primate Models for AIDS (held in Atlanta, GA, October 7-10, 1998) (abstract 79).
Showing the most recent 10 out of 492 publications
