An ordered pathway of assembly of components required for polyadenylation site recognition and processing.

Abstract

Four HeLa cell nuclear factors that are required for specific pre-mRNA cleavage and polyadenylation have been extensively purified, thereby permitting an investigation of the role of each in the 3' processing reaction. Two factors, termed PF1 and PF2, are required for specific polyadenylation of the cleaved RNA. PFI is a poly(A) polymerase, and PF2 is a factor that confers AAUAAA specificity to the reaction. Both of these factors, along with two additional factors termed CF1 and CF2, are required for the endonucleolytic cleavage of the premRNA. The ability of each of these factors to form specific complexes with the pre-mRNA was assayed using native gel electrophoresis. Two distinct complexes were detected. PF2 forms an initial complex with the pre-RNA, dependent on the AAUAAA sequence element but independent of specific downstream sequences. Formation of the PF2-RNA complex permits the subsequent interaction of CF1 and the formation of a second, larger complex. CF1 binding requires the downstream sequence element in addition to PF2 binding. Whereas the PF2-RNA complex is unstable and dissociates rapidly, the ternary complex formed by CF1, PF2, and RNA is stable. Thus, the interaction of CFI, dependent on the downstream sequence element, can be viewed as a commitment of the poly(A) site for processing. On the addition of the poly(A) polymerase (PFI) and factor CF2, the pre-mRNA is specifically cleaved at the poly(A) site.

ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts

“…Fig. 2 also shows that the DE150 fraction from the first step in the purification of polyadenylation factors (Gilmartin and Nevins, 1989) is able to use ADP efficiently for poly(A) addition to a probe containing the AAUAAA sequence element (lanes I), but not the mutated AACAAA sequence element (lanes 2). In contrast, the DE150 fraction catalyzes the ATP-supported polyadenylation of the AAUAAA probe only weakly (Fig.…”

“…Lack of a specific ATP-dependent poly(A) addition reaction in the DE150 fraction (McDevitt et al, 1988;Gilmartin and Nevins, 1989), or in similar first-step purification fractions (Christofori and Keller, 1988;Takagaki et al 1988;Bardwell et al, 1990) has been reported previously. It is well established that the poly(A) addition reaction assayed with ATP requires a non-specific poly(A) polymerase (Christofori and Keller, 1989;Gilmartin and Nevins, 1989;Bardwell et al, 1990) and at least one, and possibly several, additional factors which recognize the AAUAAA sequence element (Christofori and Keller, 1988;Takagaki et al, 1988;Gilmartin and Nevins, 1989). To test if the ADP-dependent poly(A) addition reaction also requires multiple components, the crude nuclear extract was fractionated on a 15-35% glycerol gradient and the fractions were recombined and assayed for ADP-and ATP-supported polyadenylation.…”

“…2 show that the DEAE-Sepharose 150 mM KCI fraction (DEl50) of the nuclear extract (Gilmartin and Nevins, 1989) also catalyzes higher rates of poly(A) addition with ADP than with ATP. DE150 is the fraction obtained from the first purification step of the polyadenylation factors (McDevitt et al, 1988;Gilmartin and Nevins, 1989). It contains a non-specific poly(A) polymerase (Gilmartin and Nevins, 1989), but is de- pleted of polyadenylation factor, PF 2, which recognizes the AAUAAA signal element in the ATP-dependent poly(A) addition reaction (Gilmartin and Nevins, 1989).…”

“…Polyadenylation of primary transcripts requires at least two steps : (a) a specific cleavage of the elongated transcript at a site downstream from the specific polyadenylation sequence element (AAUAAA) (Fitzgerald and Shenk, 1981 ; Gil and Proudfott, 1984;Hart et al, 1985;Zarkower et al, 1986); and (b) subsequent 3' poly(A) addition (Moore and Sharp, 1985;Zarkower et al, 1986). These reactions have been partially resolved and shown to require, in addition to a nonspecific poly(A) polymerase, a polymerase specificity factor (or factors) which recognizes the AAUAAA sequence element (Christofori and Keller, 1988;McDevitt et al, 1988;Takagaki et al, 1988;Gilmartin and Nevins, 1989), and one or more factors responsible for the cleavage process. The total number of factors required for the entire cleavage/poly(A) addition reaction is not known.…”

“…Samples were stored at -80°C. The 150 mM KC1 DEAE-Sepharose fraction (DEl50 fraction) was prepared as described (Gilmartin and Nevins, 1989). …”