Remodeling of ribonucleoprotein complexes with DExH/D RNA helicases

Abstract

The DExH/D protein family is the largest group of enzymes in eukaryotic RNA metabolism. DExH/D proteins are mainly known for their ability to unwind RNA duplexes in an ATP-dependent fashion. However, it has become clear in recent years that these DExH/D RNA helicases are also involved in the ATP-dependent remodeling of RNA-protein complexes. Here we review recent studies that highlight physiological roles of DExH/D proteins in the displacement of proteins from RNA. We further discuss work with simple RNA-protein complexes in vitro, which illuminates mechanisms by which DExH/D proteins remove proteins from RNA. Although we are only beginning to understand how DExH/D proteins remodel RNA-protein complexes, these studies have shown that an 'RNA helicase' does not per se require cofactors to displace proteins from RNA, that protein displacement does not necessarily involve RNA duplex unwinding, and that not all DExH/D proteins are able to disassemble the same range of ribonucleoproteins.

Figure 1

DExH/D proteins targeting RNA–protein interactions: physiological examples. (A) Displacement of the U1snRNP from an RNA containing a 5′ splice site by Prp28p. (B) Displacement of Mud2p by Sub2p. (C) ATP-dependent displacement of Cus2p by Prp5p. The second step shows the ATP-independent binding of U2 snRNP to the branch site of the pre-mRNA, which is facilitated by Prp5 in an ATP-independent fashion. (D) Displacement of Mex67 by Dbp5p. Dbp5p is bound to the nuclear pore complex on the cytoplasmic side of the nuclear rim. As mRNAs are transported through the nuclear pore, Dbp5p specifically facilitates the release of Mex67.

Figure 2

Model systems for measuring RNP remodeling by DExH/D proteins in vitro. (A) U1A-based RNP. Ovals indicate U1A, lines the RNA, and arrows represent the reactions catalyzed by the DExH/D protein. RNA–protein complex and free RNA is visualized on non-denaturing PAGE. (B) Trap-based RNP. RNA–protein complex and free RNA is visualized on non-denaturing PAGE. (C) EJC-based RNP. The RNA contains specific radiolabel (asterisk) in the EJC-binding region. Removal of the EJC renders the region protected previously from nuclease digestion by the EJC susceptible to degradation. Degradation was visualized on denaturing PAGE. (D) Removal of the U1 snRNP from a radiolabeled (asterisk) RNA with a 5′ splice site. Removal of the U1snRNP was monitored by immunoprecipitation of U1snRNP and subsequent quantification of radioactivity in supernatant and immunoprecipitate.

Figure 3

Possible mechanism for active protein displacement by DExH/D proteins. (A) Schematic representations for the displacement of a single (homopolymeric) protein from RNA. The line represents the RNA. (B) Schematic representations for the displacement of a multi-component protein complex from RNA.