Discovery of an element in the terminal exon of the mouse beta-globin gene that influences the accumulation of spliced RNA
This study describes how a simple transcription unit, mouse $\beta$-globin (MBG), behaves when placed in the context of a complex transcription unit. RNAse protection assays were performed, using total RNA derived from COS-1 cells transfected with a series of plasmids composed of the SV40 promoter, entire kanamycin resistance gene (neo), the 3$\sp\prime$ end of the M$\beta$G second exon, the entire second intron (modified by insertion of a Xhol linker), and terminal exon (with 3$\sp\prime$ flanking sequence) (pNG series). Modifications include insertion of an additional poly(A) site within the intron, 3$\sp\prime$SS deletion, and wild type poly(A) site deletion. Experiments show that (1) the intronic poly(A) site is essentially ignored; most transcripts are spliced and polyadenylated at the wild type poly(A) site, and (2) when splicing is prevented by deletion of the 3$\sp\prime$ splice site, use of the intronic poly(A) site predominates over use of the wild type site, but there is a drastic decrease in steady state RNA accumulation relative to control levels, and (3) removal of the wild type poly(A) site improves use of the intronic site, but RNA accumulation remains very low. This study also outlines the discovery of an element, located in a 99 nt segment of the terminal exon, that influences the accumulation of spliced RNA. Results of experiments using the pKD series of constructs (based on pNG with truncation of the intron and deletion of 99 nt from the terminal exon) show that (1) the presence of the element is absolutely required for the demonstration of spliced transcripts (unspliced, polyadenylated transcripts are demonstrable), (2) partial ("half") deletions of the element leads to a large decrease in the levels of spliced transcripts with concomitant increase in the levels of unspliced transcripts, (3) the element probably contains dyad symmetry (inversion of the 99 nt segment leads to near normal levels of spliced transcripts), (4) removal of the first of two imperfect inverted repeats leads to a moderate, but significant decrease in the level of spliced transcripts, and (5) the element appears to function by strengthening a weak polypyrimidine tract (strengthening the tract partially overcomes the necessity for the element, but optimal processing occurs only when the element is present).
Dowling, Patricia Kaye, "Discovery of an element in the terminal exon of the mouse beta-globin gene that influences the accumulation of spliced RNA" (1994). ETD Collection for Fordham University. AAI9416665.