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Chen et al. demonstrate a new way by which non-coding RNAs tailor the function of multi-component complexes. They show that a non-coding RNA interacts with an exoribonuclease, altering its substrate specificity and enzymatic activity by serving as a ribonucleoprotein scaffold, and perhaps a gate for entry of the RNA substrate.
Multi-protein complexes are the workhorses of the cell, and provide critical functions necessary for cellular growth and viability by merging related activities into compact molecular machines. Protein-protein interactions are well known to be involved in allosteric regulation, altering substrate specificity and localization of enzymatic function to specific subcellular compartments. Several RNAs that serve as scaffolds for such molecular machines have been described, including yeast TLC1 RNA and telomerase (Lebo and Zappulla, 2012), pRNA and the Ø29 DNA packaging motor (Harjes et al., 2012) and IRES elements and translation factors. The ability of RNAs to scaffold molecular machines is also being investigated for synthetic biology applications (Delebecque et al., 2012). Given the abundance of non-coding RNAs (ncRNAs) in cells, could they act as dynamic scaffolds for ribonucleoprotein (RNP) complex formation, altering enzymatic functions and regulating cellular processes? Chen et al. (2013) find that this may be the case for the Ro protein-Y RNA complex.
The authors demonstrate that in the extremophile Deinococcus radiodurans, the non-coding Y RNA acts as an adaptor between the Ro protein ortholog Rsr (an RNA binding protein) and the polynucleotide phosphorylase (PNPase, a 3′-5′ exoribonuclease) to produce an RNA degradation machine with altered substrate preference and enzymatic function (Figure 1). Previous data from the Wolin laboratory has shown that Rsr associates with Y RNA, and that interactions between Rsr and the PNPase are important for RNA degradation (Wurtmann and Wolin, 2010). How this process actually works, however, has been unclear. Based on biochemical analyses, electron microscopic image reconstruction and modeling to a known Ro protein-Y-RNA fragment, the authors present a new model in which a dual ring structure channels RNA substrates into the PNPase enzymatic cavity. In the EM reconstructions based on previous crystallographic structures, Y RNA fits within a narrow density between Rsr and the PNPase. Interestingly, no contacts between the proteins are observed. This, along with the analysis of the interactions of purified factors, suggests that Y RNA is responsible for holding the complex together. Y RNA not only serves as the backbone of the complex but also blocks the KH/S1 domain of PNPase, reducing the enzyme’s ability to interact with single-stranded RNA substrates. Thus the Rsr-Y RNA-PNPase machine is more active on structured RNA substrates and less active against single-stranded RNA substrates than the free PNPase enzyme. Interestingly, this Y RNA-assembled degradation machine appears to be conserved in Salmonella typhimurium, indicating that the components are part of an evolutionarily conserved RNP system.
In addition to the PNPase regulation described above, ribonuclease activities in cells generally appear to be tightly regulated in macromolecular complexes. The dual endo/exoribonucleases J1 and J2 in B. subtilis form a complex that regulates their enzymatic activity and substrate specificity (Mathy et al., 2010). In eukaryotic cells, the poly(A)-specific exonucleases CCR4 and CAF1 localize together in a complex that is assembled around the NOT1 scaffold (Petit et al., 2012). Rrp6 and isoforms of the Dis3 3′-5′ exonucleases are sequestered and function as part of a large exosome complex (Drazkowska et al., 2013). As these macromolecular complexes utilize protein-protein interactions to regulate ribonuclease function, it is particularly interesting that, in this example, an RNA that would ultimately be a substrate for the enzyme has been chosen by the cell to regulate the function of a powerful ribonuclease.
A major implication of the Chen et al. study is that enzymatic function/protein associations can be dynamically controlled by the level and type of the ncRNA. Most organisms contain more than one Y RNA species with a Ro protein-binding stem and significant variations in their loop structures (Sim and Wolin, 2011). Interestingly, it is the loop structures that serve as the assembly site for the PNPase, perhaps suggesting that additional proteins may be regulated in a similar fashion. Moreover, there is no a priori reason why other ncRNAs could not function in a similar fashion in other RNP machines. Thus, ncRNAs could be used to select different protein pairings and provide altered RNP functions. Indeed, direct protein-protein interactions may be only a small part of the puzzle for how environmentally responsive macromolecular machines are formed and regulated. Y RNAs, for example, are known to interact with at least five other proteins (RoBP1, hnRNP I, hnRNP K, nucleolin and ZBP1), and it will be interesting to see if such RNA-protein interactions also affects these cellular factors.
Another intriguing hypothesis is that alterations in environmental conditions might change ncRNA expression, folding or general availability, and drive the formation of RNP complexes with enhanced properties to help the cell adapt to its new environment. Along these lines, we note that while Rsr interacts with RNase II and RNase PH during heat stress to help mature rRNA (Chen et al., 2007), it interacts with the PNPase during the stationary phase to degrade misfolded RNA (Wurtmann and Wolin, 2010). It will be interesting to see if changes in scaffolding ncRNAs under these different conditions allow Rsr to form new RNP structures or alter the subcellular localization of an RNP.
In closing, this study emphasized the potential for ncRNAs to adapt protein modules to varied functions. The components of RNPs, therefore, may be easily changed by mixing and matching different parts, making them more like the beloved classic ‘Mr. Potato Head’ toy than previously thought.
B.J.G. is supported by NIH grant U54 AI065357. J.W. is supported by NIH grants R01 GM072481 and U54 AI065357.
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