Cofactor-dependent specificity of a DEAD-box protein PNAS PLUS

Crystal L. Younga,b,1, Sohail Khoshnevisb, and Katrin Karbsteinb,2

aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055; and bDepartment of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458

Edited by Alan M. Lambowitz, The University of Texas at Austin, Austin, TX, and approved April 4, 2013 (received for review February 9, 2013) DEAD-box proteins, a large class of RNA-dependent ATPases, reg- Ded1 activity, and therefore renders this system unsuitable for ulate all aspects of gene expression and RNA . They testing cofactor effects on substrate specificity (19). In contrast can facilitate dissociation of RNA duplexes and remodeling of to Ded1 and eIF4A, DEAD-box proteins involved in RNA–protein complexes, serve as ATP-dependent RNA-binding pro- biogenesis are likely to encounter specific substrates. Enzymes teins, or even anneal duplexes. These proteins have highly con- involved in ribosome assembly may therefore be more suitable served sequence elements that are contained within two RecA-like for testing the hypothesis that cofactors regulate the specificity domains; consequently, their structures are nearly identical. Further- of DEAD-box proteins. more, crystal structures of DEAD-box proteins with bound RNA re- Here, we present a biochemical characterization of the DEAD- veal interactions exclusively between the protein and the RNA box protein Rok1 and its cofactor Rrp5, an RNA-binding protein backbone. Together, these findings suggest that DEAD-box proteins that recognizes sequences in the pre-rRNA between 18S and interact with their substrates in a nonspecific manner, which is con- 5.8S rRNAs (39). Both proteins are required for early, nucleolar firmed in biochemical experiments. Nevertheless, this contrasts with 40S ribosome maturation steps (40, 41). In vitro pull-down ex- the need to target these enzymes to specific substrates in vivo. periments demonstrate that Rrp5 directly binds Rok1, consistent Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both with previously described genetic interactions (42). Rrp5 is function during maturation of the small ribosomal subunit, we bound near a pre-rRNA duplex that undergoes a conformational show here that Rrp5 provides specificity to the otherwise nonspe- change to regulate cleavage steps during ribosome assembly (39, cific biochemical activities of theRok1DEAD-domain.Thisfind- 43). Using RNA strands that mimic this duplex, we show that ing could reconcile the need for specific substrate binding of Rok1 binds the double-stranded duplex ∼20-fold stronger than fi some DEAD-box proteins with their nonspeci c binding surface either of the single-stranded substrates. We also demonstrate BIOCHEMISTRY and expands the potential roles of cofactors to specificity factors. that Rok1 stabilizes duplexes and catalyzes strand annealing, Identification of cofactors and their RNA substrates could whereas RNA duplex unwinding activity is not observed. Rok1 therefore help define the undescribed roles of the 19 DEAD-box alone binds and anneals the test duplex with low specificity rela- proteins that function in ribosome assembly. tive to other duplexes. Addition of Rrp5 enhances Rok1 anneal- ing of this duplex ∼10-fold, although essentially not affecting RNA | annealing control duplexes. This increased specificity is a result of changes in the Rok1 structure induced by Rrp5 binding. This finding EAD-box proteins are RNA-binding ATPases that are in- expands the role of DEAD-box cofactors from regulators to Dvolved in all aspects of RNA metabolism: translation initia- specificity factors while explaining how DEAD-box proteins, tion, pre-mRNA splicing, mRNA export and decay, and ribosome which lack sequence-specific RNA binding abilities in their core, biogenesis. In these processes, their functions include RNA duplex can derive the specificity for their substrates in vivo. unwinding, RNA–protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1–5). Results In vivo, these enzymes have specific functions that often in- Rok1 Directly Binds the C-Terminal Fragment of Rrp5. Previous data volve the recognition of specific and the discrimination implicate the RNA-binding protein Rrp5 as a likely cofactor for against a myriad of nonsubstrates. Nevertheless, only DbpA, a the DEAD-box protein Rok1, and Rok1 overexpression sup- DEAD-box protein involved in bacterial ribosome assembly (6–8), presses the temperature-sensitive phenotype of a mutation in the has sequence-specific ATPase activity (6, 9), which arises from first tetratricopeptide (TPR) motif of Rrp5 (42). TPR motifs are unique sequences in its C terminus (10). Such sequence specificity common protein–protein interaction modules (44), suggesting has not been demonstrated for any other DEAD-box protein and that this genetic interaction could reflect a direct interaction. is consistent with their highly conserved structures and the almost Consistent with this hypothesis, Rok1 requires the presence of universal conservation of residues that contact the RNA. Fur- Rrp5 to associate with pre-rRNA during ribosome assembly (45). thermore, analysis of the structures of RNA-bound DEAD-box To verify that Rrp5 binds Rok1, as expected for a cofactor, we proteins reveals that RNA contacts are made with the sugar- carried out an in vitro protein binding assay. Rrp5 is a 192-kDa phosphate backbone (11–17), largely precluding sequence-specific protein containing 12 S1 RNA-binding domains toward its N interactions between DEAD-box proteins and RNA. terminus and seven TPR motifs toward its C terminus. Because Many DEAD-box and related DEAH-box proteins function in expression and purification of full-length Rrp5 do not produce conjunction with cofactors (ref. 18 and references therein). These large yields of protein (39), and because the N and C termini of cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNA- binding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19–25). We and others therefore Author contributions: C.L.Y., S.K., and K.K. designed research; C.L.Y., S.K., and K.K. per- postulated that these cofactors could also function to provide formed research; S.K. contributed new reagents/analytic tools; C.L.Y., S.K., and K.K. ana- specificity for DEAD-box and DEAH-box proteins (1, 18). lyzed data; and C.L.Y. and K.K. wrote the paper. Ded1 and eIF4A, two of the best-studied DEAD-box proteins The authors declare no conflict of interest. (19, 24, 26–38), have known binding cofactors: Gle1 and eIF4G, This article is a PNAS Direct Submission. respectively. Because Ded1 and eIF4A both function in trans- 1Present address: Department of Chemistry, The College of Wooster, Wooster, OH 44691. lation initiation, a process that requires the recognition of many 2To whom correspondence should be addressed. E-mail: [email protected]. fi diverse mRNAs, sequence speci city for these particular DEAD- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. box proteins may be undesirable. Furthermore, Gle1 inhibits 1073/pnas.1302577110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302577110 PNAS Early Edition | 1of9 Downloaded by guest on September 23, 2021 3' Rrp5 can function independently (39, 42, 46), we used the G G A C-terminal fragment of Rrp5, called Rrp5C hereafter (39), for in ABA C 5' A ACA vitro protein binding experiments to test for a direct interaction A U U G G U with Rok1. This fragment contains the last 3 S1 RNA-binding C G U GA A2 G domains as well as all seven TPR motifs, and when combined with G U A U A U the N-terminal piece, it fully complements the Rrp5 deletion (39). A A UC U A When maltose-binding protein (MBP)-tagged Rrp5C is incu- C U A A bated with Rok1 and amylose beads, Rok1 is retained on the AG C U AG U G U beads (Fig. 1). In contrast, no retention on the beads is observed in A U U G G C the presence of MBP alone, indicating that Rok1 directly binds C G G C C G Bound Fraction Rrp5C; Rrp5 is therefore a binding partner for Rok1. 5' 3'

Rok1 Binds Pre-rRNA with Low Specificity. Northern blot analysis H44 ITS1 Strand St ran d [Rok1] (µM) of rRNA processing phenotypes during ribosome maturation pre-A 2 showed that Rrp5 is required for production of small and large Duplex ribosomal subunits (41, 47). Consistently, Rrp5 binds within in- ternal transcribed spacer 1 (ITS1), the intron-like sequence be- C tween 18S and 5.8S sequences in rRNA precursors (39). Within ITS1, Rrp5 binds adjacent to a dynamic, imperfect duplex (39, 43) (SI Appendix, Fig. S1). This duplex is formed early in assembly between one strand of helix 44 (H44; the decoding site helix in mature 18S rRNA) and sequences within ITS1. After cleavage at the so-called “site A2” within ITS1 (SI Appendix, Fig. S1), this duplex is replaced with the mature decoding site H44. Because this duplex is dynamic during assembly and is proximal to the

Rrp5 binding site, and because our results validated Rrp5 as an Duplexed Fraction interacting partner of Rok1, we postulated that Rok1 is involved in modulating the dynamics of this helix, referred to hereafter as the pre-A duplex. We therefore designed RNA substrates for 2 [H44] (nM) Rok1 to mimic the pre-A2 duplex, now composed of the strand

of H44 that is involved in the duplex interaction (H44) and the Fig. 2. Rok1 binds and stabilizes the pre-A2 duplex. (A) Pre-A2 duplex is strand in ITS1 (ITS1). To increase the thermodynamic stability based on Rok1’s predicted in vivo substrate (SI Appendix, Fig. S1). Four al- of the duplex, it was necessary to add four alternating G-C base ternating G-C base pairs (bold) were added to the base of the duplex to ● pairs to the base of the duplex (Fig. 2A). improve its thermodynamic stability. (B) Rok1 binds the pre-A2 duplex ( ) Control experiments were carried out to test for proper forma- with higher affinity than the ITS1 (■) or H44 (◆) ssRNAs. RNA concen- 32 SI Appendix trations were 0.5 nM P-ITS1 and 20 nM H44 for the pre-A2 duplex, and tion of duplexes between the H44 and ITS1 strands ( , 32 32 Fig. S2A). These experiments indicate that the ITS1 strand self- 0.5 nM P-ITS1 or P-H44 for ssRNAs. Fitting to Eq. 1 gives K1/2 values of 0.14 μM, 3.4 μM, and 3.6 μM for the pre-A2 duplex, ITS1, and H44 strands, anneals at high concentrations; this RNA is therefore kept trace respectively. (C) Fraction of duplex formed at 0.5 nM 32P-ITS1 and varying in all our experiments. Next, we used gel-shift analysis with H44 concentrations was quantified from gel-shift experiments. Observed K 32 1/2 P-labeled RNA substrates to compare binding of Rok1 to single- values are 65.6 nM and 2.4 nM in the absence (●)andpresence(■)of400nM stranded H44 and ITS1 and double-stranded pre-A2 duplex. As Rok1, respectively. shown in Fig. 2B, Rok1 binds the duplex ∼20-fold more tightly than either of the single-stranded RNAs (ssRNAs; K1/2 is 0.17 μM for the duplex and 2.6 μMand4.1μM for the ITS1 and H44 reverse complement of the pre-A2 duplex as well as another con- strands, respectively; these are averages from at least three experi- trol duplex (U3+27). The U3+27 duplex contains 10 Watson– ments). This finding suggests that Rok1 recognizes structures Crick base pairs and is formed between a fragment of the small within this complex double-stranded substrate. nucleolar RNA (snoRNA) U3 and its complementary binding re- ′ To confirm that the pre-A2 duplex studied here is indeed a gion within the pre-rRNA; this duplex also includes a 27-nt 3 - specific target for Rok1, we next compared Rok1’saffinity for the overhang. Duplex formation controls for the reverse complement and U3+27 duplexes are shown in SI Appendix,Fig.S2B and C, respectively. SI Appendix,Fig.S3indicates that Rok1 has low se- quence preference for the pre-A2 duplex compared with the con- trol duplexes, with average K1/2 values of 0.17 μM, 0.14 μM, and (MBP)-Rrp5C Rok1 μ Rok1 0.27 Mforthepre-A2, reverse complement, and U3+27 duplexes, Rok1 MBP respectively (these averages are from at least three experiments). IN FTWINFTEL EL WINFTELW Rok1 Stabilizes the Pre-A2 Duplex. Because Rok1 preferentially binds dsRNA, thermodynamic laws of energy conservation * maintain that Rok1 binding must similarly stabilize duplex for- mation. To test this prediction, we measured duplex formation in * the presence or absence of 400 nM Rok1 using gel-shift analysis. As predicted, the data in Fig. 2C indicate that Rok1 stabilizes the Fig. 1. Rok1 binds (MBP)-Rrp5C. Rok1 binds an MBP-tagged C-terminal formation of the pre-A2 duplex ∼30-fold (K1/2 values of 65.6 nM fragment of Rrp5 that contains the TPR motifs and three S1 RNA-binding and 2.4 nM for the absence and presence of Rok1, respectively). domains (39, 42, 46). Control experiments indicate that Rok1 does not bind to the amylose resin or the MBP tag. The asterisk indicates contaminating This enhancement agrees quantitatively with the 20-fold binding MBP and Rrp5C from the (MBP)-Rrp5C protein preparation. EL, elution; FT, preference for dsRNA observed in the gel-shift experiments and flow-through; IN, input; W, third wash. demonstrates the robustness of our system.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1302577110 Young et al. Downloaded by guest on September 23, 2021 Rok1 Promotes Annealing but Not Unwinding. Because some contacts both strands of the duplex, it is expected to stabilize the PNAS PLUS DEAD-box proteins have the ability to unwind a short RNA duplex, thereby helping to explain the annealing activity. duplex, DEAD-box proteins are often referred to as RNA heli- As with Ded1 and Mss116 (31, 32), ATP, but not ADP, inhibits cases. Nevertheless, only a third of yeast DEAD-box proteins Rok1 annealing (SI Appendix,Fig.S5). Importantly, even with have been tested for unwinding activity (26, 29, 31, 48–54), and saturating ATP, the annealing rate constant was higher than several are not efficient unwinding enzymes (21, 55). In contrast, that observed in the absence of Rok1 and the end point in some have the ability to promote duplex formation and to dis- helix formation was not affected. These data suggest that ATP sociate RNA–protein complexes or function as ATP-dependent changes the structure of Rok1, as observed with other DEAD- RNA-binding proteins (56–59). The ability to unwind duplexes is box proteins, where ATP binding leads to closure of the two thought to arise from the exclusion of dsRNAs when the two RecA domains (33, 60, 61; reviewed in refs. 1, 3, 5). Because RecA domains are closed upon each other (33, 60, 61; reviewed dsRNA cannot be accommodated in this conformation, annealing in refs. 1, 3, 5). Considering Rok1’s preference for dsRNA over activity is lost. Importantly, inhibition experiments with ADP ′ β γ ssRNA and its ability to stabilize RNA duplexes, we tested or the nonhydrolyzable ATP analog adenosine 5 -( , -imido) ’ triphosphate (AMPPNP) demonstrate that Rok1 binds ADP at Rok1 s ability to promote annealing of an RNA duplex. Pulse- SI Appendix chase gel-shift experiments were used to determine annealing least 500-fold more strongly than AMPPNP (or ATP; , rate constants for the U3+27 duplex in the presence and absence Fig. S6). Because cellular ATP concentrations are only about of Rok1 (Fig. 3A). These data reveal that Rok1 enhances duplex twofold higher than ADP concentrations (62), these data suggest that Rok1 is bound to ADP in vivo, rendering this annealing formation ∼10-fold (Fig. 3B). Similar enhancement of duplex activity relevant. formation is observed for the pre-A duplex (Fig. 3D) and the 2 To test if the observed annealing activity is specific to Rok1 or reverse complement duplex (Fig. 4). This ATP-independent – SI Appen- simply due to the special nature of this duplex rich in non-Watson annealing activity is Rok1 concentration-dependent ( Crick base pairs, we tested if Dbp8 also promoted annealing of this dix, Fig. S4A), and gives a K1/2 of 0.12 μM for the pre-A2 duplex, K μ duplex. Like Rok1, Dbp8 is a DEAD-box protein involved in early very similar to the apparent 1/2 of 0.17 M for Rok1 binding to steps of 40S ribosome maturation (63). Dbp8-dependent annealing the pre-A2 duplex obtained from RNA-binding experiments. This activity for the pre-A2 duplex was reduced >200-fold relative to agreement further demonstrates the robustness of our system and Rok1-dependent annealing activity (SI Appendix,Fig.S7A). In suggests that Rok1 functions by binding the duplex. contrast, annealing of the reverse complement was similar for both To test if the helicase core promotes annealing or whether this proteins, indicating that the Dbp8 protein is active (SI Appendix, BIOCHEMISTRY activity arises from N- and/or C-terminal extensions, we pro- Fig. S7B). Together these data provide evidence that annealing of duced the Rok1 helicase core and tested it for duplex annealing the pre-A2 duplex is specifictoRok1. activity (SI Appendix, Fig. S4 C and D). Importantly, the Rok1 To test if Rok1 has the ability to unwind duplexes, we added core alone promotes duplex annealing, albeit at higher concen- ATP and MgCl2. However, no unwinding of the U3+27 duplex trations, consistent with weaker RNA binding of the core alone. (Fig. 3C) or the longer pre-A2 and pre-A2full duplexes (SI Ap- These data demonstrate that annealing is an activity of the Rok1 pendix, Fig. S8) was observed, even in the presence of an un- helicase core, consistent with the observation that other DEAD-box labeled trap strand that can bind to the released strand. proteins, such as Ded1 and Mss116, also have annealing activity Similarly, Uhlenbeck and colleagues (55) have not found sig- (31, 32). It has been shown recently that the second RecA domain nificant unwinding activity for Rok1. It is possible that unwinding (RecA2) of Mss116 can bind dsRNA by itself (60). Because RecA2 could be observed with a specific substrate, although that is

rol A nt C 10 min 10 min co 10 min 10 min 10 min time time time time time time preannealedchase control chasepreannealed control preannealedchase

* * * * 0 nM Rok1 600 nM Rok1 600 nM Rok1 0 nM Rok1 360 nM Rok1 625 nM Rrp5C 0 nM Rrp5C 625 nM Rrp5C

BD44 ylno 1S ylno 1 µM

H H

+ 1S 0 µM

TI [Rok1] T

I*

* • Rok1 *

* Fraction Duplexed Fraction *

time (min) RNA Rok1 Rok1 Controls Anneals Binds

Fig. 3. Rok1 catalyzes duplex annealing but not unwinding. (A) Pulse-chase gel-shift analysis under protein-denaturing conditions shows that Rok1 increases the rate of duplex formation for the U3+27 duplex. The lane labeled “chase control” shows how much duplex is formed when the chase is added before duplex formation. The lane labeled “preannealed” shows how much duplex maximally forms under the conditions of the pulse. (B) Quantitation of the data − − in A yields annealing rate constants of 0.12 min 1 and 0.78 min 1 in the absence (○) and presence (●) of Rok1, respectively. (C) Rok1 does not promote unwinding of the U3+27 duplex. The preannealed duplex is added to Rok1 in the presence of ATP and trap helix. Controls show duplex before addition of Rok1 (preannealed) and the premixed trap and duplex (chase control). (D) Native gel analysis of Rok1 annealing and binding activities indicates that Rok1

anneals the pre-A2 duplex at concentrations below those required for stable binding. Rok1 concentrations are 0, 10, 25, 50, 100, 200, 400, 600, 800, and 1000 nM. RNA controls include 32P-ITS1 alone (lane 1) and an unduplexed 32P-ITS1 and H44 mixture before 10-fold dilution into reactions containing Rok1 (lane 2). Final RNA concentrations are 0.5 nM 32P-ITS1 and 20 nM H44.

Young et al. PNAS Early Edition | 3of9 Downloaded by guest on September 23, 2021 A parison of duplex formation over time in the presence of Rrp5C indicates that Rrp5C enhances the annealing activity of Rok1 ∼10-fold for the pre-A2 duplex [Fig. 4 (circles) and Table 1; kobs − − values of >0.19 s 1 and 0.022 s 1 for the presence and absence of Rrp5C, respectively]. [The annealing rate of the pre-A2 duplex in the presence of Rrp5C is too fast to be measured accurately by hand (Fig. 4A). The observed rate constant is therefore an un- derestimate as indicated by the arrow in Fig. 4B.] Rrp5C alone has no effect on the annealing rate constant (SI Appendix,Fig.S9A).

fraction annealed Furthermore, addition of Tsr1 or Fap7, other ribosome assembly factors with pI values similar to Rrp5C, does not affect Rok1- time (s) dependent annealing (SI Appendix,Fig.S9B), demonstrating that B this effect is specific for the Rok1 cofactor. In contrast, Rrp5C 0.20 enhances the annealing rate of the reverse complement duplex

) 0.04 k -1 less than twofold [Fig. 4 (squares) and Table 1; obs values of 0.016 ) 0.15 − −

-1 1 1 (sec 0.02 s and 0.01 s for the presence and absence of Rrp5C, re- obs

0.10 k 0.00 (sec pre-A reverse U3+27 spectively]. Similarly, Rrp5C provided no increase in the annealing Wild-type2 Reverse U3/37mer duplex complement duplex B k obs Duplex Control Duplex Duplex 0.05 duplex rate constant for the U3+27 duplex (Fig. 4 ; obs values of 0.0003 k −1 −1 0.00 s and 0.001 s for the presence and absence of Rrp5C, re- pre-A reverse U3+27 spectively). Therefore, the presence of Rrp5C increases the Wild-type2 Reverse Control U3/37mer duplex complement duplex specificity for Rok1 annealing of the pre-A duplex from ap- Duplex Duplexduplex Duplex 2 proximately twofold to ∼20-fold. Fig. 4. Rrp5 enhances Rok1’s annealing activity and specificity. (A) Com- Rrp5C Enhances Specificity by Changing the Rok1 Structure. The data parison of Rok1 (400 nM) annealing activities with the pre-A2 duplex (○,●) or its reverse complement (□,■) in the absence (open symbols) or presence thus far indicate that Rrp5C increases the specificity and rate of −1 (closed symbols) of 2 μM Rrp5C. Observed rate constants are 0.02 s and Rok1-dependent annealing of the pre-A2 duplex. It could do so −1 0.30 s for the pre-A2 duplex in the absence and presence of Rrp5, re- by prelocalizing Rok1 to its RNA substrate, thereby increasing its − − spectively, and 0.0039 s 1 and 0.0082 s 1 for the reverse complement duplex effective concentration (i.e., affinity), or by altering the structure in the absence and presence of Rrp5, respectively. RNA concentrations were 32 32 of Rok1 to make it more effective in annealing. To distinguish 0.5 nM P-ITS1 and 20 nM H44 for the pre-A2 duplex or 0.5 nM P-ITS1rev between these possibilities, we compared the effect of Rok1 on and 2 nM H44rev for its reverse complement. (B) Comparison of Rok1 annealing of the pre-A2 duplex and its reverse complement under annealing rate constants for the pre-A2 duplex, the reverse complement duplex, and the U3+27 duplex in the absence (black) and presence (gray) of conditions where Rok1 is saturating and subsaturating with re- 2 μM Rrp5C. Control experiments in which the concentration of Rrp5 was spect to RNA (Table 1). If Rrp5C increases Rok1 affinity by varied indicate that this concentration is saturating. The arrow above the bar prelocalizing it to the RNA, no effect from Rrp5C is expected at representing the rate constant for annealing of the pre-A2 duplex in the high Rok1 concentrations, because it is already saturating. How- presence of Rrp5 indicates that this observed rate constant is an un- ever, even at saturating Rok1 concentrations, Rrp5C increases derestimation, because the reaction was too fast to be measured accurately Rok1’s activity and specificity, suggesting that Rrp5C does not by hand (Fig. 4A). work by increasing Rok1’saffinity for RNA.* To further rule out the possibility that Rrp5C functions by generally not the case for DEAD-box proteins with helicase linking Rok1 to the RNA, we determined the Rrp5C concentra- activity. More generally, it appears that not every DEAD-box tion dependence for the provided increase in annealing rate. This K ≤ SI protein has unwinding activity (21, 55). Similarly, the closely concentration dependence saturates with a 1/2 of 30 nM ( Appendix K ∼ μ related RIG-I subclass of superfamily 2 (SF2) “helicases” seems ,Fig.S10), which is much lower than the 1/2 of 8 M to lack unwinding activity (64). observed for binding of Rrp5C to RNA (39). Furthermore, co- Because Rok1 annealing was observed at low Rok1 concen- operative binding of Rok1 and RNA to Rrp5C, which could in- fi SI Appendix trations, under which Rok1 is not stably bound to duplexes, we crease the Rrp5C af nity for RNA, is not observed ( , wondered if Rok1 catalyzes duplex formation without stably bind- Fig. S11). Together, these data suggest that Rrp5C can increase ing to duplexes. To test this idea, we modified the annealing ex- annealing of Rok1 without binding to RNA, strongly indicating periment by omitting the SDS in the gel-loading buffer, reasoning that it functions by changing the Rok1 structure. that this would allow us to monitor both Rok1 annealing and RNA To test directly if Rrp5C changes Rok1 structure, we carried binding. Fig. 3D shows that at low Rok1 concentrations (10–200 out limited proteolysis of Rok1 in the presence and absence of nM), duplex formation, but not stable RNA binding, is observed. In Rrp5 (Fig. 5). Thermolysin produced different proteolytic frag- A contrast, stable RNA binding requires Rok1 concentrations ≥400 ments for Rok1 alone and in complex with Rrp5 (Fig. 5 ). To confirm that these are Rok1 and not Rrp5 fragments, we probed nM. These results indicate that Rok1 is actively catalyzing pre-A2 duplex formation in a manner that could allow for turnover, pro- the proteolytic digests with antibodies directed against Rok1 B vided that the duplex is stable after Rok1 dissociates. and Rrp5 (Fig. 5 ). As expected, if Rrp5 changes the Rok1 structure, these data show that the presence of Rrp5 leads to Rrp5C Enhances Rok1 Annealing and Specificity. To determine if Rok1 alone has specificity for the pre-A2 duplex, we compared the annealing rate for this duplex with that observed with the *To ensure that the saturation of the annealing activity at high concentrations of Rok1 was due to binding of Rok1 and not a change in the rate-limiting step, we determined reverse complement control duplex. Rok1 anneals the pre-A2 the Rok1 concentration dependence in the presence and absence of Rrp5C. If a change duplex approximately twofold faster than the reverse comple- in the rate-limiting step was responsible for the apparent saturation behavior, both ment duplex (Fig. 4A, open circles) and approximately fivefold concentration dependencies should saturate with the same plateau but with about − 1 10-fold lower K values in the presence of Rrp5C. In contrast, the data in SI Appendix, faster than the U3+27 duplex (Fig. 4B, Inset; kobs values of 0.01 s , 1/2 − − 1 1 Fig. S4 A and B show that Rrp5C increases the plateau and provides a slightly higher K1/2 0.005 s , and 0.0019 s for annealing of the pre-A2, reverse value (K1/2 = 0.13 μM and 0.38 μM in the absence and presence of Rrp5C, respectively). complement, and U3+27 duplexes, respectively). Next, we tested These data strongly suggest that saturation of Rok1 arises from binding to its substrate, the effect of Rrp5C on the annealing activity of Rok1. Com- and thus provide support for the idea that Rrp5C functions by changing Rok1 structure.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1302577110 Young et al. Downloaded by guest on September 23, 2021 Table 1. Rrp5 enhances Rok1 annealing in a sequence-specific manner PNAS PLUS

Pre-A2 duplex Reverse complement duplex

Subsaturating Rok1 Saturating Rok1 Subsaturating Rok1 Saturating Rok1

− − − − −Rrp5C 0.01 ± 0.002 s 1 0.022 ± 0.002 s 1 0.005 ± 0.002 s 1 0.01 ± 0.007 s 1 +Rrp5C 0.044 ± 0.026 s−1 ≥0.19 ± 0.03 s−1 0.005 ± 0.004 s−1 0.016 ± 0.009 s−1 Fold rate enhancement 4.4 9.5 1.0 1.6

All experiments were carried out at 20 °C in the presence of 40 mM Tris (pH 8.3), 100 mM KCl, and 2 mM DTT. All data are averages from two to four independent experiments. The rate constant for annealing in the presence of saturating Rok1 and Rrp5C is too fast to − be measured accurately by hand (Fig. 4A). Thus, the provided rate constant of 0.19 s 1 is likely an underestimation of the true rate constant, as indicated by the “≥” symbol.

the appearance of new proteolysis fragments in Rok1 and also Here, we demonstrate that Rrp5 is a cofactor of the DEAD- protects Rok1. Time courses confirm that these fragments are box protein Rok1, thereby extending the short list of DEAD-box formed preferentially in the presence of Rrp5 and are not simply proteins for which cofactors have been identified. Importantly, more stable in the presence of Rrp5 (Fig. 5C). Finally, the con- Rrp5 increases Rok1 substrate specificity ∼10-fold, thereby ex- centration dependencies for Rok1-dependent annealing in the tending the roles of cofactors from simple regulators to speci- presence and absence of Rrp5C suggest that Rrp5 binding slightly ficity factors, as previously hypothesized (1, 18). Our data also weakens Rok1’s RNA binding, because these saturate with three- demonstrate that Rrp5 can provide this effect without binding to RNA, and regardless of whether Rok1 is saturating or sub- fold different K1/2 values (SI Appendix,Fig.S4A and B; K1/2 = 1.3 μMand3.8μM, respectively). Importantly, this difference saturating; this suggests that Rrp5 changes the Rok1 structure, as fi in the RNA binding affinity also provides evidence for changes con rmed in limited proteolysis experiments, perhaps by expos- fi in Rok1’s RNA binding pocket up on binding of Rrp5. ing a new RNA binding surface that provides sequence speci city. This is reminiscent of recent observations made with other heli- Discussion cases. Gle1 changes the structure of the DEAD-box protein Dbp5,

Expanding the Role of Cofactors for DEAD-Box Proteins. Of the 25 thereby remodeling its RNA binding site (66). Similar conforma- BIOCHEMISTRY DEAD-box proteins in Saccharomyces cerevisiae, five (eIF4A, Fal1, tional changes have been previously observed on binding of eIF4G Ded1, Dbp5, and Dbp8) are known to be regulated by cofactors to eIF4A (28, 73), and they have also been described for the SF1 (12, 19, 21, 28, 34, 35, 65–67). Similarly, three related SF2-heli- helicase Upf1 up on binding of its cofactor Upf2 (74). In these cases, it remains to be seen if these changes affect the RNA- cases from the DEAH and Ski2-like subfamilies (Prp43, Mtr4, and binding specificity, even though the interaction with eIF4G Brr2) are also regulated by cofactors (22, 23, 25, 68–71). These increases specificity for unwinding of helices with a 5′-overhang by cofactors can increase or decrease the rates of ATP hydrolysis, eIF4A (75). This role of cofactors in improving the specificity of RNA unwinding, or phosphate or RNA release. Furthermore, fi DEAD-box proteins via allosteric changes in the DEAD-box cofactors can also increase or decrease nucleotide or RNA af n- proteins augments the previously postulated role of recruitment ities (reviewed in ref. 18). Cofactors therefore contribute to the of helicases to their substrates, which requires the prebinding of temporal regulation of the activities of DEAD-box proteins. the cofactor to its substrate via RNA binding, or in the case of In addition to being activated at the appropriate time, DEAD- Nup159, via binding to the correct position on the nuclear pore. box proteins must be activated in response to the correct sub- How general is the model in which substrate specificity arises strate. This is not trivial, because DEAD-box proteins recognize from interactions with cofactors? There is only one documented the RNA backbone, therefore precluding strong sequence case where a DEAD-box protein has substantial intrinsic speci- specificity (11–17). Cofactors are believed to contribute to the ficity. DbpA, a bacterial RNA helicase required for 50S ribo- spatial regulation of these helicases by recruiting them to their some maturation, has ATPase activity that is specifically targets, which have cofactors already bound; for example, activated by helix 92 in 23S rRNA (7, 9, 10). For DbpA, speci- Nup159 is located to the cytoplasmic face of the nuclear pore, ficity arises from its C-terminal extension (10). Most DEAD-box where it is believed to promote Dbp5-dependent remodeling of proteins have N- and/or C-terminal extensions outside of the RNA–protein complexes after nuclear export (12, 66, 72). helicase core that could therefore provide specificity in a similar

Fig. 5. Binding of Rrp5 changes Rok1 structure. (A) Coomassie-stained SDS/PAGE of Rok1, Rrp5, and Rok1–Rrp5 complex treated with thermolysin. Regions that differ between complex and individual proteins are marked with an arrowhead. (B) Western blot analysis of the same proteolytic digests as in A.(C) Rok1 Western blot analysis of proteolysis time courses. The control lane (C) contains the Rrp5·Rok1 complex in the absence of protease.

Young et al. PNAS Early Edition | 5of9 Downloaded by guest on September 23, 2021 manner. However, no such specificity has been observed for any the bound RNA, which leads to unsticking, and thus weakens the studied helicase (ref. 1 and references therein), including those duplex (11, 14), and steric exclusion of one strand, when RecA1 whose substrates are well-delineated from genetic studies; this docks onto the duplex bound to RecA2 (60). RNA bending is suggests that our observation that cofactors can mediate speci- stabilized by interactions with structures formed by the con- ficity for DEAD-box proteins could be widespread. Interestingly, served motifs 1b and 1c (11, 14), but it also appears to be forced it has recently been demonstrated that eIF4G confers specificity by a loop on the opposite side of the RNA strand, which, oth- for a 5′-overhang toward the helicase activity of eIF4A (75), and erwise, would be in steric conflict with the RNA (shown in red in it has been speculated that this structural specificity could be SI Appendix, Fig. S12). Interestingly, the structure of RecA1 important in the directional movement of initiating 40S ribo- from human Rok1 demonstrates that this loop is significantly somes from the cap toward the start codon. shorter, and therefore cannot contribute to RNA bending (SI Like DEAD-box proteins, DEAH-box proteins are part of the Appendix, Fig. S12). Furthermore, the steric clash with the op- SF2 family of RNA helicases. The DEAH-box protein Prp43 has posite RNA strand that arises from the loop C-terminal to the roles in pre-mRNA splicing as well as ribosome biogenesis (22, helix containing the DEAD-motif (shown in black in SI Appen- 76–78). Interestingly, these roles are mediated by different dix, Fig. S12) is reduced. We speculate that these two features cofactors (22, 23, 25, 71, 79), suggesting that cofactor-mediated together might contribute to the lack of unwinding activity ob- specificity could even hold true for other members of the SF2 served in Rok1, and perhaps other DEAD-box proteins. helicases besides DEAD-box proteins. Furthermore, in ribosome assembly, Prp43 has two different cofactors: Gno1 and Pfa1. Roles of Rrp5·Rok1 in Ribosome Assembly. Genetic (42) and bio- Prp43 has been implicated in the removal of snoRNAs from chemical (45) data suggested that the RNA-binding protein Rrp5 pre-rRNA (77, 78, 80, 81). It is tempting to speculate that one could be an interacting partner for the DEAD-box protein Rok1. of the cofactors provides specificity for box C/D snoRNAs, We have tested this hypothesis here and demonstrate that Rrp5 whereas the other provides specificity for box H/ACA snoRNAs. indeed binds to Rok1. Furthermore, Rok1 promotes the formation These snoRNAs differ by their conserved namesake sequence of a duplex we have termed the pre-A2 duplex both kinetically elements, their bound proteins, and the type of modification to as well as thermodynamically. This duplex is formed between rRNA that they encode (82–84). one strand of the decoding site helix, H44, and an RNA sequence “ ” The example of Prp43 also illustrates a possible advantage of downstream of so-called cleavage site A2 early in assembly and SI Appendix encoding specificity via an external factor: The resulting modularity is disrupted after A2 cleavage (43) ( ,Fig.S1). Inter- allows the same RNA helicase to be used in multiple processes, estingly, this conformational switch is required for subsequent therefore facilitating evolution. Furthermore, the modularity of formation of the 3′-end of 18S rRNA. We therefore initially the process allows for regulation of the two processes either speculated that Rok1 was involved in mediating the disruption of independently (via the cofactors) or together (via the helicase). this helix after A2 cleavage. The data here, however, suggest that Nineteen DEAD-box and DEAH-box proteins are required this is not the case for several reasons. First, Rok1·Rrp5C has fi for ribosome assembly in yeast (1). Despite extensive efforts, annealing activity that is speci c for the pre-A2 duplex. In contrast, their substrates and roles during assembly of the two ribosomal no unwinding activity was observed for any tested duplex (55). This subunits remain largely unknown. The data presented here indicates that Rok1·Rrp5 is unlikely to be involved in duplex un- suggest that the solution to uncovering helicase function may be winding. Furthermore, in the absence of Rok1, the pre-A2 duplex is to identify their cofactors first. By uncovering the RNA-binding not very stable; this indicates that such an unwinding activity may sites for helicase·cofactor complexes, substrates for helicases can not be necessary. Finally, depletion of Rok1 or Rrp5 disrupts ri- i be delineated for further study. bosome assembly before A2 cleavage (40, 41, 46). Given that ( )the Rok1·Rrp5 complex has specific annealing activity for the pre-A2 Cofactors Confer Specificity in Other Biological Processes. Here, we duplex, (ii) this inhibitory duplex is formed between two RNA show that a cofactor can enhance the specificity of a DEAD-box strands that are >300 nt away from each other in comparison to protein. The concept of cofactors conferring substrate specificity is the local H44 sequences, and (iii) Rrp5·Rok1 stabilize this duplex, not unique to DEAD-box proteins, likely reflecting the evolu- we speculate that Rrp5·Rok1 could be involved in the formation tionary advantages discussed above. For example, cofactor-me- of this duplex early in assembly, consistent with effects on rRNA diated specificity is also observed during , where the processing. However, testing this hypothesis is beyond the scope general transcription machinery is directed to individual pro- of this work and will require future in vivo experiments. moters by transcription factors, which bind DNA in a sequence- dependent manner (85, 86). Similarly, the activity and substrate Materials and Methods specificity of cyclin-dependent kinases are regulated by the cyclin Cloning of Rok1. The Rok1 ORF was amplified from S. cerevisiae genomic DNA 84–517 they bind (87). Further, during ubiquitination, the E1 and E2 and cloned between the SfoI and HindIII sites of pSV272. Rok1 was enzymes obtain substrate specificity via the E3-ligase (88). cloned into pGEX-6P-1 containing an N-terminal GST tag (GE Healthcare). Primers are listed in SI Appendix, Table S1.

How Do DEAD-Box Proteins Promote Annealing? Rok1 can promote fi fi annealing without stably binding to the ssRNA substrate. In- Rrp5C and Rok1 Expression and Puri cation. Rrp5C was puri ed as described K K (39), and for (MBP-)Rrp5C tobacco etch virus (TEV) protease was omitted and stead, the 1/2 for annealing is similar to the d for binding of dialysis was carried out in 50 mM NaCl, 25 mM Hepes (pH 7.6), 1 mM Tris dsRNA, indicating that Rok1 recognizes and transiently sta- (2-carboxyethyl)phosphine (TCEP), and 1 mM DTT. MBP-Rrp5C concentration bilizes the first base pairs of a forming duplex, thereby increasing was determined by absorbance at 280 nm using a calculated extinction co- − − the probability that duplex formation completes instead of dis- efficient of 128,160 M 1·cm 1. For overexpression of Rok1, Escherichia coli sociation of the initial base pairs. Rosetta2 (DE3) cells containing the Rok1-encoding pSV272 plasmid were RecA2 of Mss116 binds dsRNA independent of the first RecA1 grown in LB/Miller medium (supplemented with 25 μg/mL kanamycin and 34 μ ∼ (60). Because RecA2 interacts with both strands, and there- g/mL chloramphenicol, respectively) at 37 °C to an OD600 of 0.6 before β fore preferentially binds duplex RNA, it is expected to stabilize inducing with 1 mM isopropyl- -D-thiogalactopyranoside in the presence of 2% (vol/vol) ethanol at 18 °C for ∼18 h. Cells were resuspended in lysis buffer duplexes, thus providing a molecular explanation for the ob- [50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF, served annealing activity of Mss116. It therefore seems likely that and 5 mM benzamidine] and sonicated. After pelleting, the soluble fraction many DEAD-box proteins have some annealing activity. In was purified over Ni-NTA resin (Qiagen) according to the manufacturer’s contrast, not all DEAD-box proteins appear to have unwinding protocol. Rok1-containing elution fractions were pooled and further puri-

activity (21, 55). Unwinding is thought to arise from bending of fied by precipitation of contaminants with 35% (vol/vol) 3.9 M (NH4)2SO4,

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1302577110 Young et al. Downloaded by guest on September 23, 2021 PNAS PLUS followed by precipitation of Rok1 with 50% (NH4)2SO4. Rok1 was resus- ITS1 was incubated with varying H44 concentrations in the presence of 400 pended in elution buffer [50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 250 nM Rok1. The fraction of RNA duplexed vs. H44 concentration was plotted,

mM imidazole] and dialyzed overnight in 200 mM NaCl, 50 mM Tris (pH 7.8), and data were fit to Eq. 1 to determine K1/2. 2 mM EDTA, 1 mM TCEP, and 1 mM DTT; TEV protease was also added to fi remove the His6-MBP tag. Rok1 was further puri ed over a MonoS column Rok1 Annealing and Unwinding Experiments. Annealing and unwinding (GE Healthcare) in a linear gradient from 440 to 720 mM NaCl over 12 col- experiments were modified from the work of Jankowsky and Putnam (92). fi umn volumes in the presence of 2 mM EDTA, followed by gel ltration using For annealing reactions, Rok1 was preincubated for 5 min at 20 °C in HRB a Superdex200 column (GE Healthcare) in 150 mM KCl, 50 mM Hepes (pH buffer. Annealing was started by the addition of the two RNA strands [10 7.6), 1 mM DTT, and 1 mM TCEP. Rok1 was stored in 15% glycerol at −80 °C. mM MOPS (pH 6.5), 1 mM EDTA, and 50 mM KCl]. For pulse-chase experi- Protein concentration was determined by absorbance at 280 nm using − − ments, labeled U3 was added to Rok1 and the unlabeled 37mer in HRB a calculated extinction coefficient of 23,980 M 1·cm 1. To determine the buffer was incubated for varying amounts of time before a 10-fold excess of activity and integrity of the Rok1 protein, we measured the ATPase activity unlabeled U3 chase was added. Control experiments established that the of WT Rok1 and the K172A mutant in the Walker A motif. At 5 μM, WT Rok1 − chase was effective and that enough 37mer was added to allow for stable hydrolyzes ATP with a rate constant of 0.4 min 1, whereas the rate constant for ATP hydrolysis by the K172A mutant was 100-fold reduced to 0.0044 duplex formation. For unwinding reactions, Rok1 was preincubated in HRB − min 1 (SI Appendix, Fig. S13). These data support the notion that the puri- buffer with 2 mM ATP/MgCl2 and a 10-fold excess of unlabeled U3. Reactions – fied Rok1 is active and well-folded. Rok184 517 was expressed in E. coli strain were initiated by the addition of U3+27 duplex. Reactions were quenched Rosetta2 (DE3) cells in 2× yeast tryptone (YT) medium. Cell lysis and binding into an equal volume of 50 mM EDTA, 1% SDS, and 0.1% bromophenol blue of the GST-tagged protein to the GSH-Sepharose resin (GE Healthcare) were in 20% glycerol and stored on ice before loading on a 15% native acryl- done in buffer A [500 mM NaCl, 50 mM Tris·HCl (pH 7.5), 5% glycerol, and 2 amide/THEM gel. Gels were dried and exposed to a phosphor screen, and mM β-mercaptoethanol (β-ME)]. The resin was washed with buffer B (same as data were quantified and fit to Eq. 2 to determine kobs: buffer A but with 300 mM NaCl), and the protein was eluted in buffer C [150 ¼ max − max • ð − • Þ: mM NaCl, 50 mM Tris·HCl (pH 7.5), 5% glycerol, 2 mM β-ME, and 20 mM fractionannealed fractionannealed fractionannealed exp kobs t [2] reduced glutathione]. The GST tag was removed using PreScission protease To determine the K for Rok1, annealing experiments were carried out (GE Healthcare). The GST tag, noncleaved protein, and protease were re- 1/2 at varying Rok1 concentrations and k values vs. Rok1 concentrations moved using an SP Sepharose column (GE Healthcare). The protein was obs fi fi further purified on a Superdex S-75 size-exclusion chromatography column were quanti ed and twithEq.3: (GE Healthcare) in 200 mM NaCl, 10 mM Hepes (pH 7.5), and 1 mM DTT. ½ n ¼ kobs;max protein : kobs n [3] ½protein þ K = RNA Transcription, Purification, and Labeling. Small RNAs were chemically 1 2

synthesized (IDT). The 37mer RNA was in vitro-transcribed using T7 RNA BIOCHEMISTRY To determine the K1/2 for Rrp5C, annealing experiments were carried out polymerase and a synthetic DNA duplex composed of a primer containing with 25 nM Rok1 and varying Rrp5C concentrations. The resulting kobs values the T7 promoter and a complementary oligo encoding the T7 promoter were plotted vs. Rrp5C concentrations and fit with Eq. 3. sequence, followed by the sequence of the 37mer RNA (T7 promoter and To determine the effects of ATP and ADP on Rok1’s annealing activity, 400 T7-RNA, respectively; SI Appendix, Table S1). Primers were mixed and nM Rok1 was preincubated at 20 °C for 5 min in the presence of increasing denatured at 95 °C for 1 min before incubating at room temperature for at least 10 min to allow primers to anneal fully. In vitro transcription was concentrations of either ADP/MgCl2 or ATP/MgCl2. Annealing reactions were 32 conducted as previously described (89, 90) using 300 nM template. Synthetic initiated by the addition of 0.5 nM P-ITS1 and 20 nM H44. To determine KI and in vitro-transcribed RNAs were purified on a 15% or 10% native acryl- values for ATP and ADP, rate constants were plotted against nucleotide amide gel, respectively, in TBE buffer (50 mM Tris, 40 mM boric acid, and concentration and fit to Eq. 4: 0.5 mM EDTA). RNA was visualized by UV shadowing, excised from the gel, kmax eluted overnight in TE buffer [10 mM Tris (pH 8.0) and 1 mM EDTA] and ¼ annealing : kannealing ½ [4] precipitated, and concentration was determined by absorbance at 260 nm. 1 þ nucleotide KI RNA was 5′-end-labeled as described (18). To analyze Rok1 annealing and binding under conditions in which protein Rok1 and Rrp5C Interaction. Rok1 (5 μM) and (MBP)-Rrp5C (or MBP; 2.5 μM) remained native, reactions were prepared as described above for annealing were mixed in binding buffer [100 mM KCl and 30 mM Hepes (pH 7.6)] and conditions, except that the SDS was omitted from the quench and reactions preincubated on ice for 15 min before addition of 15 μLofequilibratedam- were immediately separated on a 10% native acrylamide/THEM gel. ylose resin (New England BioLabs). The mixture was incubated on a rotating platform at 4 °C for 30 min before flow-through fractions were collected. Resin Rok1 ATPase Inhibition Experiments. Rok1 (1 μM) was incubated at 20 °C for was then washed four times with 55 μL of binding buffer before being eluted 5 min in the presence of 40 mM Tris (pH 8.3), 50 mM KCl, 2 mM DTT, 10 mM μ with 30 L of binding buffer supplemented with 50 mM maltose. MgCl2, and increasing concentrations of either ADP or the nonhydrolyzable ATP analog AMPPNP. For concentrations of AMPPNP greater than 1 mM, an

RNA Binding Experiments. RNA binding experiments were carried out as equimolar amount of MgCl2 was added. After preincubating at 20 °C for 32 described (18, 39). For binding to dsRNA, 5 nM P-labeled RNA was pre- 5 min, ATPase reactions were initiated by the addition of purified 32P-γ-ATP. incubated at 95 °C for 1 min with enough unlabeled RNA to give full duplex Reaction mixtures were quenched at various time points with an equal volume formation (exact concentrations are provided in figure legends) in the of 0.75 M KH2PO4 (pH 3.3). Quenched reaction mixtures were spotted onto presence of 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 6.5), a polyethyleneimine (PEI) nitrocellulose TLC plate and eluted in 1 M LiCl, 300 1 mM EDTA, and 50 mM KCl. RNAs were slowly cooled to 35 °C and then mM NaH2PO4 (pH 3.8). TLC plates were exposed to a phosphor screen, and the incubated on ice for 10 min. RNA was diluted 10-fold into preincubated 32 32 amount of P-γ-ATP and Pi was determined using phosphorimager soft- (20 °C) reaction mixtures containing Rok1 in the presence of HRB buffer ware. The fraction of Pi was plotted as a function of time, and data were fitto [40 mM Tris (pH 8.3), 50 mM KCl, 0.5 mM MgCl , and 2 mM DTT]. Reactions 2 Eq. 1. To determine K values, observed rate constants were plotted against were incubated at 20 °C for 7 min before being separated on a 10% native I nucleotide concentration and the data were fittoEq.4. acrylamide/THEM [Tris, Hepes, EDTA (pH 7.5), and MgCl2] gel (91). Control experiments in which the incubation time was varied from 2 to 20 min in- dicated that this was sufficient for equilibration. Gels were exposed to a Limited Proteolysis. Thermolysin (Roche) was incubated with Rok1, Rrp5, or an phosphor screen, quantified using phosphorimager software, and analyzed equimolar mixture of Rok1 and Rrp5 in a ratio of 1:100 (wt/wt) in a buffer as described (39, 90). The dependence of the fraction of Rok1-bound RNA containing 130 mM NaCl and 30 mM Mes (pH 6.8) at room temperature. In all μ on the Rok1 concentration was fit to Eq. 1: experiments, the protein concentrations were set at 6 M. After 20 min (or varying times), the reaction was stopped by the addition of SDS loading dye 2 – fraction ; ½protein and samples were analyzed by SDS/PAGE on 4 15% precast Mini-Protean TGX fraction ¼ bound max : [1] bound 2 gels (Bio-Rad) for the single time point experiment or 12% SDS gels for the time ½protein þ K1=2 courses. To detect changes in the proteolytic pattern of individual proteins, To measure duplex stability in the presence and absence of Rok1, an- samples were analyzed by Western blotting using anti-Rok1 (93) and anti-Rrp5 nealing reactions were prepared as above, except 0.5 nM of radiolabeled antibodies raised by Josman, LLC against recombinant full-length proteins.

Young et al. PNAS Early Edition | 7of9 Downloaded by guest on September 23, 2021 ACKNOWLEDGMENTS. We thank E. Jankowsky and R. Russell for helpful This work was supported by National Institutes of Health Grant R01-GM086451 discussions and members of our laboratory for comments on the manuscript. and a beginning investigator grant from the American Heart Association.

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