Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation.

Interactions required for inhibition of poly(A) polymerase (PAP) by the Ul snRNP-specifi c UlA protein, a reaction whose function is to autoregulate UlA protein production, are examined. PAP inhibition requires a substrate RNA to which at least two molecules of UlA protein can bind tightly, but we demonstrate that the secondary structure of the RNA is not highly constrained. A mutational analysis reveals that the carboxy-terminal 20 amino acids of PAP are essential for its inhibition by the UlA-RNA complex. Remarkably, transfer of these amino acids to yeast PAP, which is otherwise not affected by UlA protein, is sufficient to confer UlA-mediated inhibition onto the yeast enzyme. A glutathione S-transferase fusion protein containing only these 20 PAP residues can interact in vitro with an RNA-UIA protein complex containing two UlA molecules, but not with one containing a single UlA protein, explaining the requirement for two UlA-binding sites on the autoregulatory RNA element. A mutational analysis of the UlA protein demonstrates that amino acids 103-119 are required for PAP inhibition. A monomeric synthetic peptide consisting of the conserved UlA amino acids from this region has no detectable effect on PAP activity. However, the same UlA peptide, when conjugated to BSA, inhibits vertebrate PAP. In addition to this activity, the UlA peptide-BSA conjugate specifically uncouples splicing and 3'-end formation in vitro without affecting uncoupled splicing or 3'-end cleavage efficiencies. This suggests that the carboxy-terminal region of PAP with which it interacts is involved not only in UlA autoregulation but also in the coupling of splicing and 3'-end formation.