Achieving Specific RNA Cleavage Activity by an Inactive Splicing Endonuclease Subunit Through Engineered Oligomerization

Abstract

Protein-protein interaction is a common strategy exploited by enzymes to control substrate specificity and catalytic activities. RNA endonucleases, which are involved in many RNA processing and regulation processes, are prime examples of this. How the activities of RNA endonucleases are tightly controlled such that they act on specific RNA is of general interest. We demonstrate in this study that an inactive RNA splicing endonuclease subunit can be switched "on" solely by oligomerization. Furthermore, we show that the mode of assembly correlates with different RNA specificities. The recently identified splicing endonuclease homolog from Sulfolobus solfataricus, despite possessing all of the putatively catalytic residues, has no detectable RNA cleavage activity on its own but is active upon mixing with its structural subunit. Guided by the previously determined three dimensional structure of the catalytic subunit, we altered its sequence such that it could potentially self-assemble thereby enabling its catalytic activity. We present the evidence for the specific RNA cleavage activity of the engineered catalytic subunit and for its formation of a functional tetramer. We also identify a higher order oligomer species that possesses distinct RNA cleavage specificity from that of previously characterized RNA splicing endonucleases.

RNA-splicing endonuclease structure and function

“…This expectation has led to the hypothesis that the catalytic and structural units pair first to form the isologous dimers, followed by the electrostatic interactions that produce the tetramer, but this hypothesis has yet to be proven experimentally. Overall, among the structural units, only the b8-L10-b9 region is strongly conserved, consistent with its primary role in tetramerization [41,45]. These findings have established the foundation for what is believed to be the architecture common to all splicing endonucleases.…”

“…A recent study of the new (ab) 2 family of endonucleases showed that the ability of the endonucleases to recognize a broader range of substrates resides within the catalytic subunit itself. The catalytic subunit of an (ab) 2 endonuclease was engineered to resemble an a 4 endonuclease (eliminating the need for the b subunit) and was found to retain most of its native recognition properties [45] (Fig. 4b).…”

“…3). Although structural information for the fully assembled abgd and (ab) 2 endonucleases is still lacking, yeast two-hybrid studies on the yeast endonuclease subunits [9] and biochemical studies on a (ab) 2 endonuclease [43,45] also support this conserved architecture. The loop L10 of each structural unit is labeled and arrows indicate the pockets into which they insert to form the tetramerization interface.…”

“…The structure is adapted from [41]. (B) A protein engineering experiment [45] which demonstrates that the conserved loop L10 is essential to cleavage activity through its role in tetramer formation. Each catalytic unit contains a highly conserved Cterminal domain.…”

A Conserved Lysine Residue in the Crenarchaea-Specific Loop is Important for the Crenarchaeal Splicing Endonuclease Activity

“…The interaction between the L10 loop in the structural subunit and the receptor pocket in the catalytic subunit is also conserved. 33 The N-terminal subdomain, which is missing in the PAE-EndA, interacts with the opposing catalytic subunit through hydrophobic interactions from tryptophan 69, valine 73, and isoleucine 76 in α-helix 3.…”