Mammalian mRNA poly(A) tails, formation removal and function

• Forschungsstelle: EU FRP
• Projektnummer: 95.0625-2
• Projekttitel Englisch: Mammalian mRNA poly(A) tails, formation removal and function

Schlüsselwörter (Englisch)

Messenger RNA; poly(A)/ 3'-end formation; RNA processing

Alternative Projektnummern (Englisch)

EU project number: FMRXCT960096

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 10.1 Stimulation of training and mobility

Kurzbeschreibung (Englisch)

See abstract

Partner und Internationale Organisationen (Englisch)

A. Virtanen (coordinator), Uppsala, B. Clements, Glasgow, N. Proudfoot, Oxford, C. Tsiapalis, Athen, P. Vassalli, Genf, E. Wahle, Halle

Abstract (Englisch)

Generation of the mature 3'-ends of mammalian messenger RNAs requires six separable protein factors. These are cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase (PAP) and poly(A) binding protein II (PABP II).
The protein factors involved in 3'-end processing are highly conserved in evolution. Twelve of the fifteen polypeptides that comprise the yeast 3'-end processing apparatus have recognizable mammalian homologues. The yeast protein Fip1 (a component of polyadenylation factor I [PF I]) was originally identified in a two-hybrid screen for factors interacting with poly(A) polymerase. We have found several human ESTs with significant homology to yeast Fip1 and have prepared an antibody directed against a heterologously expressed human Fip1 fragment. Preliminary results indicate that human Fip1 copurifies with CPSF activity from HeLa cells.
CF I is found in several isoforms. A common 25 kD subunit is associated with one of three larger possible polypeptides of 59 kD, 68 kD or 72 kD. We have shown that the 68/25 kD form of CF I can be reconstituted in active form from recombinantly expressed polypeptides. We have cloned and expressed the 59 kD subunit and have found that this too can be reconstituted with the 25 kD polypeptide to form active CF I factor. We are now making mutants in the different subunits and we are searching for interacting proteins. We also study the interaction of the CF I isoforms with the pre-mRNA substrate.
We have further purified mammalian cleavage factor II (CF II) and have analyzed its associated polypeptides by microsequencing. Among the proteins identified are the human homologues of two 3'-end processing polypeptides from yeast, Pcf11 and Clp1, both of which are genuine subunits of yeast cleavage factor IA (CF IA). We have isolated cDNA clones that code for human Pcf11 and human Clp1 and have raised antibodies directed against heterologously expressed human Clp1 and human Pcf11. The antibodies inhibit the cleavage step of the mammalian 3'-end processing reaction.
We have made a series of mutations in mammalian poly(A) polymerase and have identified a region in the enzyme adjacent to the catalytic center that is responsible for binding the ATP substrate. This domain is conserved in many nucleotidyltransferases.
As a first step towards getting a high-resolution picture of the polyadenylation machinery, we have determined the crystal structure of mammalian poly(A) polymerase complexed to 3'-dATP in the active site. The structure provides insights to the mechanism of polyadenylation and to the substrate specificity of the enzyme. (In collaboration with Dr. Sylvie Doublié, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington VT, USA).

Datenbankreferenzen (Englisch)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 95.0625-2