The processing and accumulation of RNA from intron-dependent and intron-independent genes
Abstract
This study is concerned with how sequences from the intron-containing mouse $\beta$-globin (M$\beta$G) gene and the naturally intronless herpes simplex virus-1 thymidine kinase (TK) gene influence the processing and accumulation of mRNA. RNase mapping was used to assay total RNA from transiently transfected COS-1 cells. The M$\beta$G gene was shown to require at least one functional intron for mRNA accumulation. Deletion of intron 2 alone depressed M$\beta$G transcript accumulation more than deletion of intron 1 alone; this is due to both differences in the introns themselves, and to the spacing between the introns and two elements: a required exonic element (EE) and the M$\beta$G polyadenylation site. Also, an unpaired 5$\sp\prime$ splice-site can depress M$\beta$G mRNA accumulation even in the presence of the EE. M$\beta$G-TK chimeras were analyzed to study how intron-independent gene TK sequences influence M$\beta$G mRNA accumulation. Insertion of a 1.1kb TK gene fragment partially ameliorated the mRNA depletion seen with intronless, but not 3$\prime$ splice-site deleted, M$\beta$G transcripts. This demonstrated that the mechanism of mRNA depletion differs between these deletions. In addition, the ability of TK sequence to restore M$\beta$G accumulation was highly dependent on its position relative to the 5$\sp\prime$ end of the chimera. This is consistent with a TK sequence-dependent co-transcriptional channelling of transcripts into an intron-independent metabolic pathway. A "polyA trap" was used to answer the question of whether the low accumulation of transcripts from intronless M$\beta$G genes are due to an inhibition of 3$\sp\prime$ end processing. Data from two different sets of polyA trap constructs are most consistent with M$\beta$G 3$\sp\prime$ end processing taking place despite the absence of introns. Therefore, the low accumulation of transcripts from intronless M$\beta$G genes is not primarily due to a lack of 3$\sp\prime$ end processing.
Subject Area
Molecular biology
Recommended Citation
Bordonaro, Michael, "The processing and accumulation of RNA from intron-dependent and intron-independent genes" (1995). ETD Collection for Fordham University. AAI9530018.
https://research.library.fordham.edu/dissertations/AAI9530018
