Article pubs.acs.org/biochemistry
The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C‑Tail To Tether RNA Substrates Veronica F. Busa, Maxwell J. Rector, and Rick Russell* Department of Molecular Biosciences and Institute for Cellular & Molecular Biology, University of Texas at Austin, Austin, Texas 78712, United States S Supporting Information *
ABSTRACT: DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich “C-tail” that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or “threshold” number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.
DEAD-box helicase proteins participate in nearly all cellular processes involving RNA. They accomplish their diverse roles by using energy from ATP binding and hydrolysis to engage in a cycle of tight, yet regulated, binding to a segment of singlestranded RNA. For many DEAD-box proteins, this cycle produces local RNA unwinding that promotes rearrangements of structured RNAs or ribonucleoprotein complexes.1−4 All DEAD-box proteins include a core of two RecA-like domains with a number of conserved motifs. The core domains bind ATP and RNA and unwind short RNA helices.5−8 While the structures and activities of the core are conserved, many DEAD-box proteins also include amino- or carboxyl-terminal domains or extensions that vary widely in size and composition.9 These ancillary domains contribute to the diversity of cellular functions for individual DEAD-box proteins, in some cases by interacting with individual substrates10−12 or groups of substrates13−16 to direct DEAD-box proteins to their cellular targets. CYT-19 is a DEAD-box protein that functions as a chaperone for several group I introns in the mitochondria of Neurospora crassa.17 CYT-19 can function in folding of group I and group II introns when expressed in a strain of Saccharomyces cerevisiae that lacks the functional ortholog, Mss116,18 indicating a role as a general RNA chaperone. To achieve this general chaperone activity, CYT-19 uses its ATPdependent helicase activity to disrupt RNA structure, allowing misfolded RNAs additional opportunities to fold to the native state.13,19,20 Single-molecule fluorescence and biochemical approaches showed that CYT-19 does not directly disrupt © XXXX American Chemical Society
RNA tertiary structure but instead captures and unwinds transiently exposed helices, leading to an RNA chaperone activity that depends on the global stability of the RNA structure.21,22 The dependence on global RNA stability is suggested to bias CYT-19 toward misfolded RNA structures, which are likely to be less stable on average than their native counterparts.21,22 The conserved core of CYT-19 is flanked by a small region termed the C-tail. Eleven of 50 amino acids in the C-tail are arginine, making the region highly basic.23 Biochemical experiments showed that removing the C-tail has a minimal effect on unwinding of short RNA helices in isolation but reduces the level of unwinding of helices that are appended to a group I intron RNA or even to just an additional helix.23 In addition, small-angle X-ray scattering (SAXS) measurements showed that the C-tail is unstructured and lies adjacent to core domain 2, such that it is well positioned to interact with RNA structural elements.15 Together, these results led to a model in which the C-tail enhances RNA unwinding activity of CYT-19 by binding nonspecifically to structured RNA and positioning the helicase core for unwinding of nearby RNA helices. Binding of the C-tail to structured RNAs is also suggested to enable multiple rounds of local RNA unwinding in a single association event with structured RNA.8,21 These roles are reminiscent of Received: April 20, 2017 Revised: May 26, 2017 Published: June 26, 2017 A
DOI: 10.1021/acs.biochem.7b00362 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry Table 1. CYT-19 C-Tail Construct Names and Corresponding Sequences
*
The C-terminal 54 amino acids of the wild-type CYT-19 protein sequence are shown. For mutants, all arginines are indicated and bolded residues in mutant sequences represent changes from the wild-type sequence.
digested plasmid was treated with phosphatase. Products were isolated with a Qiagen PCR cleanup kit and then ligated using T4 DNA ligase. The entire coding sequences of the clones were then verified via Sanger sequencing. Expression and Purification of Wild-Type and Mutant CYT-19 Proteins. Proteins were expressed as fusions with an N-terminal MBP tag. Each protein was expressed and purified as described previously23 except that the tag was retained (Figure S1). We found that the presence of this tag does not impact RNA unwinding activity significantly, as the secondorder rate constants measured for MBP fusions of wild-type and Δ578−626 mutant proteins were the same within error as measured previously for the untagged proteins.23 RNA Unwinding Assays. RNA unwinding activity was measured using gel mobility shift assays essentially as described previously.13 To monitor dissociation of the substrate from the Tetrahymena ribozyme, 25 nM ribozyme was incubated for 5 min at 25 °C in the presence of 10 mM Mg2+ and 50 mM Na+MOPS at pH 7.0. This incubation gives predominantly misfolded ribozyme,29,30 which provides a defined substrate for measuring unwinding of the P1 helix.13,23 A trace amount (
