Eukaryotic mRNAs are not replicas of the genes that encode them. Instead, they are produced by a series of post- transcriptional modifications of a primary transcript. In principle, each maturation step provides a means of regulating mRNA-formation. In the past four years, using molecular biological approaches, we have identified regions of the mRNA precursor that are required for cleavage (formation of the 3' terminus), and showed that a factor binds stably to one of these regions during the reaction. We propose a model which provides the framework for many of the experiments described here. We plan an intensive analysis of three mRNA processing steps: cleavage, polyadenylation, and transport from the nucleus to the cytoplasm. We will use the mRNA of tumor virus, SV40, as a model and will assay maturation both in vivo and in vitro. Sequences essential for each step will be identified both by mutation and by direct chemical means. To further analyze each reaction, we will: (1) identify those features of each base which are critical for processing; (2) identify those phosphates which participate in processing and determine whether they do so by RNA-protein contacts; (3) identify factors required for processing and the activation of maternal mRNAs in C. elegans; (4) identify component(s) that bind stably to the precursor during cleavage; (5) test whether processing requires recognition of the 5' end of the pre-mRNA by using a circular RNA; and (6) determine what features of the precursor are necessary for transport by injecting processing intermediates into nuclei. The experiments proposed will have important practical applications. Because various organisms use similar but distinct mechanisms to process their mRNAs, the opportunity exists to create a new generation of antibiotics directed against a previously unexploited target - mRNA processing. Our work will provide fundamental knowledge for the rational design of such drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Modified Research Career Development Award (K04)
Project #
5K04GM000521-03
Application #
3072877
Study Section
Molecular Biology Study Section (MBY)
Project Start
1988-07-01
Project End
1993-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Bilger, A; Fox, C A; Wahle, E et al. (1994) Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements. Genes Dev 8:1106-16
Sheets, M D; Fox, C A; Hunt, T et al. (1994) The 3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev 8:926-38
Forrester, W; Stutz, F; Rosbash, M et al. (1992) Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure. Genes Dev 6:1914-26
Fox, C A; Sheets, M D; Wahle, E et al. (1992) Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase. EMBO J 11:5021-32
Bardwell, V J; Wickens, M; Bienroth, S et al. (1991) Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition. Cell 65:125-33
Fleischmann, M; Clark, M W; Forrester, W et al. (1991) Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation. Mol Gen Genet 227:417-23
Ogg, S C; Anderson, P; Wickens, M P (1990) Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract. Nucleic Acids Res 18:143-9
Wickens, M (1990) In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biochem Sci 15:320-4
Wickens, M (1990) How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci 15:277-81
Fox, C A; Wickens, M (1990) Poly(A) removal during oocyte maturation: a default reaction selectively prevented by specific sequences in the 3' UTR of certain maternal mRNAs. Genes Dev 4:2287-98

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