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

Samuel I. Gunderson, Stéphan Vagner, Maria Polycarpou-Schwarz, Lain W. Mattaj

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Abstract

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 UIA-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 a to regulatory 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.

Original languageEnglish (US)
Pages (from-to)761-773
Number of pages13
JournalGenes and Development
Volume11
Issue number6
DOIs
StatePublished - Mar 15 1997

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All Science Journal Classification (ASJC) codes

  • Genetics
  • Developmental Biology

Keywords

  • RNA processing
  • U1A protein
  • cleavage and polyadenylation
  • poly(A) polymerase
  • pre-mRNA splicing

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