The Effect of
Gene Localization on RNA Processing
Chao Chen and Lawrence A. Chasin
Several lines of evidence suggest that the nucleus of mammalian cells is compartmentalized. It is possible that the position of a gene either within the 3‑dimensional space of the nucleus or within a local chromatin domain could play an important role in the efficiency with which its transcripts are spliced or polyadenylated, or its mRNA is transported from the nucleus. To examine this hypothesis, we electroporated a 40 kb cosmid fragment that contained the 25 kb dhfr gene together with a pSV2gpt gene into the CHO deletion mutant DG44 which lacks the dhfr locus. Nine clones that had received a single copy of the dhfr gene were examined by fluorescence in situ hybridization (FISH). The transgenes had integrated into a different location in each case. Steady‑state dhfr mRNA levels measured by RNase protection and quantitative RT‑PCR varied from 1/5 to 3 times the level found in a CHO hemizygote carrying a single dhfr gene in its natural chromosomal location (wild type). To test the idea that dhfr transcripts were differentially processed in independent integrants, we measured the ratio of dhfr pre‑mRNA to dhfr mRNA by RT‑PCR. All of the integrants exhibited a higher ratio than wild type cells; in the most extreme case, the ratio was 20 times higher than wild type. A higher ratio was found for transfectants that exhibited steady-state mRNA levels that were either higher or lower than wild type. We interpret these results to mean that some step(s) in dhfr RNA processing was slower in the integrants. Splicing is one of the steps that may be sensitive to gene position. we therefore measured the in vivo splicing rate of selected introns by stopping RNA synthesis with Actinomycin D and compared the rate of disappearance of intron‑exon joints in 2 transfectants and the wild type. A transfectant with a lower dhfr mRNA level had a 2-fold lower splicing rate, while that with the higher mRNA level showed a slightly faster splicing rate than the wild type (which had a t 1/2 of 6 minutes). We conclude that splicing could be affected by gene position, but the 2‑fold difference observed was insufficient to account for the greater differences in pre‑mRNA:mRNA ratio. The length of dhfr poly(A) tail in the transfectants was also measured, using a poly(U)‑ Sepharose affinity spin chromatography assay and an RT‑PCR‑based assay. Similar lengths of poly(A) tails were found for the dhfr RNAs of wild type and the two transfectants studied. We conclude that gene position can influence post‑transcriptional RNA metabolism, and speculate that the major effect may be at the level of transport and/or nuclear mRNA degradation.