Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome

Abstract

We have previously demonstrated that PM-Scl-75, a component of the human exosome complex involved in RNA maturation and mRNA decay, can specifically interact with RNAs containing an AU-rich instability element. Through the analysis of a series of deletion mutants, we have now shown that a 266 amino acid fragment representing the RNase PH domain is responsible for the sequence-specific binding to AU-rich elements. Furthermore, we found that the RNase PH domains from two other exosomal components, OIP2 and RRP41, as well as from Escherichia coli polynucleotide phosphorylase, are all capable of specifically interacting with RNAs containing an AU-rich element with similar affinities. Finally, we demonstrate that the interaction of the RNase PH domain of PM-Scl-75 is readily competed by poly(U), but only inefficiently using other homopolymeric RNAs. These data demonstrate that RNase PH domains in general have an affinity for U- and AU-rich sequences, and broaden the potential role in RNA biology of proteins containing these domains.

FIGURE 1.

The RNA binding domain of PM-Scl-75 maps to the RNase PH domain. (A) Recombinant wild-type (WT) PM-Scl-75 and three deletion variants were generated as depicted. (B) Increasing amounts of recombinant PM-Scl-75 protein and each of the three variants were incubated with 5 fmol of matched RNA substrates that either contained an AU-rich element (Gem-ARE-A0) or lacked the 34 base insert (Gem-A0). A total of 20 pmol of GST were used as a negative control. RNA–protein complexes were detected by gel-shift analysis on 5% native acrylamide gels.

FIGURE 2.

(A) The PM-Scl-75 PH domain is specific for AU-rich sequences. Increasing amounts of recombinant protein were incubated with 5 pmol of Gem-A0, Gem-ARE-A0, or Gem-ARE_mt-A0 as indicated. RNA–protein complexes were detected by gel-shift analysis on 5% native gels. (B) Poly(U), but not poly(A) or poly(C), is an effective competitor of PM-Scl-75 sequence-specific RNA interaction. The indicated amount of poly(U), poly(C), or poly(A) was added as a competitor to reaction mixtures containing 6 pmol of PM-Scl-75 protein and 5 pmol of Gem-ARE-A0 RNA substrate. RNA–protein complexes were detected by gel-shift analysis on 5% native acrylamide gels.

FIGURE 3.

The RNase PH domains of OIP2, RRP41, and PNPase all possess sequence-specific RNA binding ability. A schematic representation of three of the RNase PH domain–containing components of the eukaryotic exosome (OIP2, PM-Scl-75, and RRP41) as well as PNPase from E. coli is shown in A. (B) The RNase PH domain–containing regions (black boxes in A) were prepared as N-terminal GST-fusion proteins, run on a 10% acrylamide gel, and stained with Coomassie blue to assess purity. The molecular weight ladder is indicated at the left of the gel. (C) The indicated pmol amounts of recombinant proteins containing only the RNase PH domain of PM-Scl-75, OIP2, RRP41, or N-terminal RNase PH domain of E. coli PNPase were incubated with 5 fmol of Gem-ARE-A0 RNA or a matched control (Gem-A0) transcript. GST (18 pmol) acted as a negative control. RNA–protein complexes were detected by gel-shift analysis on 5% native acrylamide gels. K_d plots are shown for each protein.