A protein interaction framework for human mRNA degradation

Abstract

The degradation of mRNA is an important regulatory step in the control of gene expression. However, mammalian RNA decay pathways remain poorly characterized. To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay. The results provide evidence for interactions between many different proteins required for mRNA decay. Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases. Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways. The interaction network also predicts the internal organization of the exosome and Lsm complexes. Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression. These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation. The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways.

Figure 1

Subunit interactions of the human exosome. (A) Y2H interactions detected on stringent (–Ade) and less-stringent (–His) selection are indicated. The amino acid residues included in protein fragments are shown in brackets. (*) Self-active bait. (B) Stringent Y2H interactions are superimposed on a model of the exosome. The Rrp41-PM/Scl-75 interaction was only detected by library screening (see Table 2). Interactions previously detected by mammalian two-hybrid (M2H) and between the homologous yeast proteins are indicated. PH-domain subunits are blue, S1-domain subunits are red, and other subunits are yellow.

Figure 1

Subunit interactions of the human exosome. (A) Y2H interactions detected on stringent (–Ade) and less-stringent (–His) selection are indicated. The amino acid residues included in protein fragments are shown in brackets. (*) Self-active bait. (B) Stringent Y2H interactions are superimposed on a model of the exosome. The Rrp41-PM/Scl-75 interaction was only detected by library screening (see Table 2). Interactions previously detected by mammalian two-hybrid (M2H) and between the homologous yeast proteins are indicated. PH-domain subunits are blue, S1-domain subunits are red, and other subunits are yellow.

Figure 2

Subunit interactions of the human Lsm complexes. (A) Each potential Lsm–Lsm interaction was assayed on selective media lacking His and containing 0 mM, 5 mM, 10 mM, 25 mM, or 50 mM 3-AT, a competitive inhibitor of the HIS3 reporter. The interactions for each Lsm bait construct are shown at the indicated concentration of 3-AT, whereby no more than two interactions are seen with proteins from either the expected Lsm1–7 or Lsm2–8 complexes. (B) These interactions are shown on models of the Lsm1–7 and Lsm2–8 complexes. The most similar Sm protein is indicated in brackets for each Lsm protein.

Figure 3

Protein–protein interactions detected between proteins predicted to function in human RNA decay. (A) Each Y2H interaction (selected on media lacking Ade or His) is indicated as an arrow from bait to prey. The amino acid residues included in protein fragments are indicated in brackets. Very weak interactions are not shown. The figure was drawn using BioLayout (http://maine.ebi.ac.uk:8000/services/biolayout/; Enright and Ouzounis 2001). (B) The interactions of the Upf proteins suggest how both nuclear and cytoplasmic 5′→3′ and 3′→5′ decay factors may be recruited during NMD. Y2H interactions are shown as arrows, proteins required for NMD are indicated by *, and proteins that coprecipitate from cell extracts are underlined (Lejeune et al. 2003).