Determinants within an 18-Amino-Acid U1A Autoregulatory Domain That Uncouple Cooperative RNA Binding, Inhibition of Polyadenylation, and Homodimerization

AUTOR(ES)

Guan, Fei

FONTE

American Society for Microbiology

RESUMO

The human U1 snRNP-specific U1A protein autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. Previous work demonstrated that a short sequence of U1A protein is essential for autoregulation and contains three distinct activities, which are (i) cooperative binding of two U1A proteins to a 50-nucleotide region of U1A pre-mRNA called polyadenylation-inhibitory element RNA, (ii) formation of a novel homodimerization surface, and (iii) inhibition of polyadenylation by inhibition of poly(A) polymerase (PAP). In this study, we purified and analyzed 11 substitution mutant proteins, each having one or two residues in this region mutated. In 5 of the 11 mutant proteins, we found that particular amino acids associate with one activity but not another, indicating that they can be uncoupled. Surprisingly, in three mutant proteins, these activities were improved upon, suggesting that U1A autoregulation is selected for suboptimal inhibitory efficiency. The effects of these mutations on autoregulatory activity in vivo were also determined. Only U1A and U170K are known to regulate nuclear polyadenylation by PAP inhibition; thus, these results will aid in determining how widespread this type of regulation is. Our molecular dissection of the consequences of conformational changes within an RNP complex presents a powerful example to those studying more complicated pre-mRNA-regulatory systems.

Documentos Relacionados

• Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation
• Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin (ST Ia).
• A complex secondary structure in U1A pre-mRNA that binds two molecules of U1A protein is required for regulation of polyadenylation.
• Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target.
• Interaction of N-terminal domain of U1A protein with an RNA stem/loop.