Related Experiment Videos
Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences
R K Hampson1, L La Follette, F M Rottman
1Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Abstract:
In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.
Insights
Sequences within exon 5 of bovine growth hormone mRNA influence the splicing of intron D. Specific elements, including an inverted repeat, regulate intron removal, impacting mature mRNA formation.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Splicing
Background:
- Alternative splicing of bovine growth hormone (bGH) mRNA can lead to incompletely processed transcripts retaining intron D.
- Previous work identified an alternatively processed bGH mRNA species lacking intron D removal.
Purpose of the Study:
- To investigate the role of downstream exon sequences in regulating the splicing of intron D in bGH mRNA.
- To identify specific sequence elements within exon 5 that influence intron D splicing.
Main Methods:
- Transient expression of the bovine growth hormone gene in Cos I cells.
- Site-directed mutagenesis involving deletions and insertions within exon 5.
- Analysis of bGH mRNA splicing patterns using Northern blotting or similar techniques.
Main Results:
- Deletion of a specific fragment in exon 5 significantly impaired intron D removal, leading to >95% of mRNA retaining the intron.
- Insertion of a 10-base-pair inverted repeat sequence partially restored normal splicing, promoting intron D removal.
- Multiple sequence elements within exon 5, not just the inverted repeat, were found to influence intron D splicing.
- The observed effects on intron D splicing were specific to the bGH gene, as exon 5 did not affect splicing of a heterologous intron (bovine prolactin intron).
Conclusions:
- Sequences within exon 5 of bGH mRNA play a critical role in regulating the splicing of the adjacent intron D.
- A unique interaction between central exon 5 sequences and intron D is proposed to govern splicing efficiency.
- These findings highlight sequence-specific regulatory mechanisms in mRNA processing.