Discriminatory RNP remodeling by the DEAD-box protein DED1

Abstract

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between al arge number of RNA and RNP complexes in ap hysiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which anon-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from at ightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the ''inability'' to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in ad iscriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

Involvement of DEAD-box Proteins in Group I and Group II Intron Splicing. Biochemical Characterization of Mss116p, ATP Hydrolysis-dependent and -independent Mechanisms, and General RNA Chaperone Activity

“…These characteristics may include: (i) ability to bind RNA and RNP substrates non-specifically; (ii) high on and off rates for RNA binding by the catalytic domain, enabling multiple cycles of activity on the same RNA; (iii) duplex unwinding without preferred polarity and ability to unwind blunt-end substrates, enabling remodeling of diverse substrates and substrate regions; (iv) limited processivity of duplex unwinding and/or an optimal balance between duplex-unwinding and strand-annealing activities to avoid over disruption of RNA structure; and (v) ability to displace or remodel incorrectly bound proteins from RNP complexes. 43,44 Finally, our findings show that while CYT-19, Mss116p, and Ded1p all have RNA chaperone activity, they differ in their ability to promote the splicing of different group I and group II intron RNAs. These differences likely reflect that the balance of RNA-binding, duplexunwinding, and strand-annealing activities has been tuned by coevolution to function optimally only on specific RNA substrates.…”

From unwinding to clamping — the DEAD box RNA helicase family

“…Proteins with larger footprints cannot be displaced by DEAD box proteins, whereas other helicases readily remove such proteins 49,53 . Although it is not known whether protein removal and duplex unwinding by DEAD box proteins rely on identical mechanisms, observations are consistent with a displacement mode that is not based on translocation of the DEAD box protein 50 . The inability to displace a certain set of proteins from RNA might be important for limiting RNA remodelling activity of DEAD box helicases in the cell to avoid complete dissociation of RNP complexes 52 .…”

“…Moreover, proteins can be displaced from structured and from unstructured RNA, suggesting that duplex unwinding is not required for release 49 . In accordance with a non-processive activit y of DEAD box proteins, protein displacement has been directly observed for proteins with a footprint of fewer than eight nucleotides [49][50][51][52] . Proteins with larger footprints cannot be displaced by DEAD box proteins, whereas other helicases readily remove such proteins 49,53 .…”

“…In addition to ATPdependent RNA unwinding and clamping, DEAD box proteins can remove proteins from RNA in an ATPdriven reaction, as has been suggested for the removal of yeast Mud2 by the DEAD box protein Sub2 during pre-mRNA splicing and for the removal of mRNA export factor 67 (Mex67) during mRNA export by DEAD box protein 5 (Dbp5) [47][48][49][50][51][52] . Moreover, proteins can be displaced from structured and from unstructured RNA, suggesting that duplex unwinding is not required for release 49 .…”

Roles of DEAD-box proteins in RNA and RNP Folding

“…Further, the discovery that some DEAD-box proteins function as general RNA chaperones in folding of the relatively simple group I and group II introns produced model systems that have allowed biochemical dissection of the roles of DEAD-box proteins snRNP from RNA in a reaction that requires disruption of both RNA-protein and RNA-RNA contacts. 136,137 DDX42, a human DEAD-box protein, can displace the single-stranded binding protein T4gp32 from ssRNA. 138 Although these activities do not necessarily reflect the in vivo functions of these proteins, they establish that remodeling RNA-protein complexes is within the catalytic repertoire of DEAD-box proteins.…”