The 3' ends of eukaryotic messenger RNAs are formed by cleavage of the primary transcript, followed by addition of a poly(A) tail. Polyadenylation is required for RNA stability and the efficiency by which a gene's transcript is polyadenylated directly influences the level to which its mRNA accumulates and is translated. This is exemplified by a case of alpha-thalassemia in which a single point mutation was found in the poly(A) signal of the alpha-globin gene. One goal of the proposed research is to define the structural requirements of poly(A) signals. The experimental approach involves mutagenesis of portions of poly(A) signals followed by functional assays involving transfected COS cells or HeLa cell nuclear extracts. The mouse beta maj globin and SV40 early poly(A) signals will be studied in detail, as well as a variety of hybrid and synthetic constructs. The concept of poly(A) signals as interrupted palindromes is used as a guide for many of the experiments. The second goal of the proposed research is to elucidate the rules that govern poly(A) site selection. How poly(A) sites are selected when other poly(A) or splicing signals are located on the transcript will be studied using both in vivo and in vitro approaches. The relationship between poly(A) signals and introns will be explored in detail. These studies will give insight into the regulation of differential RNA processing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033410-06
Application #
3283113
Study Section
Molecular Biology Study Section (MBY)
Project Start
1987-09-01
Project End
1992-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Fordham University
Department
Type
Graduate Schools
DUNS #
City
Bronx
State
NY
Country
United States
Zip Code
10458
Bordonaro, M; Nordstrom, J L (1994) Different mechanisms are responsible for the low accumulation of transcripts from intronless and 3' splice site deleted genes. Biochem Biophys Res Commun 203:128-32
Bordonaro, M; Saccomanno, C F; Nordstrom, J L (1994) An improved T1/A ribonuclease protection assay. Biotechniques 16:428-30
Saccomanno, C F; Bordonaro, M; Chen, J S et al. (1992) A faster ribonuclease protection assay. Biotechniques 13:846-50
Chen, J S; Nordstrom, J L (1992) Bipartite structure of the downstream element of the mouse beta globin (major) poly(A) signal. Nucleic Acids Res 20:2565-72
Kessler, M M; Westhafer, M A; Carson, D D et al. (1987) Polyadenylation at a cryptic site in the pBR322 portion of pSV2-neo: prevention of its utilization by the SV40 late poly(A) signal. Nucleic Acids Res 15:631-42
Kessler, M M; Beckendorf, R C; Westhafer, M A et al. (1986) Requirement of A-A-U-A-A-A and adjacent downstream sequences for SV40 early polyadenylation. Nucleic Acids Res 14:4939-52
Nordstrom, J L; Westhafer, M A (1986) Splicing and polyadenylylation at cryptic sites in RNA transcribed from pSV2-neo. Biochim Biophys Acta 867:152-62
Nordstrom, J L; Hall, S L; Kessler, M M (1985) Polyadenylylation of sea urchin histone RNA sequences in transfected COS cells. Proc Natl Acad Sci U S A 82:1094-8