Accumulation of mouse beta-globin transcripts in transfected COS cells is influenced by the identity and arrangement of pre -mRNA processing signals
Transient transfections of COS cells allowed analysis of the processing and accumulation of transcripts from pSV2neo/mouse $\beta$-globin chimera. Apparent dominance of splicing over polyadenylation was demonstrated by the functional silence of a pA signal placed within the only intron of pG- and pNG-series constructs (the wild-type pA signal was functional). Splice site deletions rendered control constructs intronless--all of which failed to accumulate readily detectable mRNA. This suggested intron-dependence of globin gene expression. Placing the SV40 t-antigen intron in an upstream position allowed terminal splice site deletions which did not render constructs intronless in either the absence (pSGB1-) or presence (pSGB2-series) of an intronic pA signal. The pA signal within the (second) intron was still functionally silent. However, profiles of transcripts from splice site deleted constructs revealed a pattern of processing and accumulation suggesting a functional link between each pA signal and its immediate-upstream 3$\sp\prime$ splice site. More native globin series were designed to facilitate serial deletions of terminal splice sites (with (pCFS2) and without (pCFS1) intronic pA signals). All deletions of the terminal 3$\sp\prime$ss were associated with mRNA accumulations of $\le$15% that of wild-type, pCFS1. Deletions of the terminal 5$\sp\prime$ss resulted exclusively in skip-splicing and 50-90% wild-type mRNA accumulation. The functional link appeared to involve the authentic terminal 3$\sp\prime$ss and the globin pA signal. Analogous p$\beta$G-series were cloned with a shortened version of intron 2 replacing intron 1. Deletion of the downstream 3$\sp\prime$ss allowed accumulation to $\le$40% that of its wild-type. Additional removal of the downstream 5$\sp\prime$ss allowed accumulation to 70-100% that of its wild-type, p$\beta$G1 (consistent with in vitro data of Niwa et al. 1992 that unpaired 5$\sp\prime$ splice sites within terminal exons depress accumulation). Moreover, the intronic pA signal is productively utilized when flanking splice sites are removed--thus revealing that the 3$\sp\prime$ss of intron 2/exon 3 is required for full use of the globin pA signal and suggesting that the overall definition of the 3$\sp\prime$ terminal exon is a major determinant of mRNA accumulation among intron-dependent eukaryotic genes.
Saccomanno, Colette F, "Accumulation of mouse beta-globin transcripts in transfected COS cells is influenced by the identity and arrangement of pre -mRNA processing signals" (1994). ETD Collection for Fordham University. AAI9416677.