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 U1 snRNP-specific U1A protein, a reaction whose function is to autoregulate U1A protein production, are examined. PAP inhibition requires a substrate RNA to which at least two molecules of U1A 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 U1A-RNA complex. Remarkably, transfer of these amino acids to yeast PAP, which is otherwise not affected by U1A protein, is sufficient to confer U1A-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-U1A protein complex containing two U1A molecules, but not with one containing a single U1A protein, explaining the requirement for two U1A-binding sites on the autoregulatory RNA element. A mutational analysis of the U1A protein demonstrates that amino acids 103-119 are required for PAP inhibition. A monomeric synthetic peptide consisting of the conserved U1A amino acids from this region has no detectable effect on PAP activity. However, the same U1A peptide, when conjugated to BSA, inhibits vertebrate PAP. In addition to this activity, the U1A 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 U1A autoregulation but also in the coupling of splicing and 3'-end formation.
Gunderson
S I
SI
European Molecular Biology Laboratory, Heidelberg, Germany.
Vagner
S
S
Polycarpou-Schwarz
M
M
Mattaj
I W
IW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity
D005656:Fungal Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D012313:RNA; D011062:Polynucleotide Adenylyltransferase