YEAST PROTEIN LINKAGE MAPS/TAPIR: Construction of protein linkage maps between yeast Saccharomyces cerevisiae nuclear proteins involved in RNA processing

• Forschungsstelle: EU FRP
• Projektnummer: 95.0136
• Projekttitel Englisch: YEAST PROTEIN LINKAGE MAPS/TAPIR: Construction of protein linkage maps between yeast Saccharomyces cerevisiae nuclear proteins involved in RNA processing

Schlüsselwörter (Englisch)

RNA ETABOLISM; NUCLEAR; TWO-HYBRID; YEAST

Alternative Projektnummern (Englisch)

EU project number: BIO4CT950009

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Kurzbeschreibung (Englisch)

See abstract

Partner und Internationale Organisationen (Englisch)

P. Legrain (coordinator) Paris, J. Beggs, Edinburgh, E. Hurt, Heidelberg, F. Lacroute, Gif-sur-Yvette, C. Marck /
M. Werner, Gif-sur-Yvette

Abstract (Englisch)

3'-end processing of yeast messenger RNA precursors (pre-mRNA) requires four biochemically separable factors. Cleavage of the pre-mRNA substrate occurs in the presence of cleavage factors I and II (CF I and CF II) and specific polyadenylation requires CF I, polyadenylation factor I (PF I) and poly(A) polymerase. CF I can be separated into two components, CF IA and CF IB. CF IA is a terameric protein complex containing Rna14p, Pcf11p, Clp1p and Rna15p as subunits. The major poly(A)-binding protein Pab1p copurifies with CF IA and acts in the control of the length of the poly(A) tails. CF IB consists of a single polypeptide encoded by the HRP1/NAB4 gene; the factor is responsible for specifying the correct cleavage site. PF I contains nine polypeptides and the genes coding for these subunits have been identified; they include homologues of all four subunits of the mammalian cleavage and polyadenylation specificity factor CPSF (Yhh1/Cft1p, Ydh1/Cft2p, Ysh1p and Yth1p), poly(A) polymerase, Pta1p, Fip1p, and two new gene products, Pfs1p and Pfs2p. We have developed a one-step purification procedure to isolate PF I by affinity purification with a protease-cleavable protein A-tagged version of the Pfs2 gene. PF I purified this way contains all nine previously identified subunits. These include the four polypeptides present in purified CF II. Accordingly, PF I is active in the cleavage as well as in the polyadenylation step of the reaction. Thus, the CF II factor appears to arise as a subcomplex that separates from PF I upon column chromatography. We propose to call the intact PF I/CF II holoenzyme complex cleavage and polyadenylation factor (CPF). We have further characterized the Pfs2p subunit of CPF biochemically and genetically. Pfs2p contains seven WD40 repeats and is required for both steps of the reaction, endonucleolytic cleavage as well as polyadenylation and the WD40 repeats are necessary for the function of the protein. GST-pulldown experiments and co-immunoprecipitation has shown that Pfs2p makes protein-protein contacts with Rna14p, Ysh1p and Fip1p. Because Rna14p is a component of CF I we propose that Pfs2p forms a bridge between CF I and CPF in the 3-'end processing complex.
We have established an extensive protein-protein interaction map between the components of the pre-mRNA 3'-end processing apparatus and other yeast proteins with the two-hybrid screening procedure. One of the interesting new proteins identified is Pti1p which interacts with the CPF subunit Pta1p. Temperature-sensitive mutants of Pti1p have no defect in pre-mRNA 3'end processing. However, ts mutant cells accumulate polyadenylated mRNAs in the nucleus after a shift to the non-permissive temperature. The Pti1 protein is associated with the nuclear pores. Thus, Pti1p may be a factor that couples 3'-end processing to mRNA export.

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: 95.0136