D-box

Subscriber access provided by Kaohsiung Medical University

Article

Substrates determinants for unwinding activity of the DExH/D-box protein RNA helicase A Qingyun Xie, Jing Liu, Yanke Shan, Shouyu Wang, and Fei Liu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01025 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Substrates determinants for unwinding activity of the DExH/D-box protein RNA helicase A Qingyun Xie1, Jing Liu1, Yanke Shan1, Shouyu Wang1,2 and Fei Liu1, * 1

MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu

Engineering Laboratory of Animal Immunology, Single Molecule Nanometry Laboratory (Sinmolab), College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China 2 Computational

Optics Laboratory, School of Science, Jiangnan University, Wuxi, Jiangsu

214122, China

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

RNA helicase A (RHA) as a member of the DExH/D-box subgroup of helicase superfamily II, is involved in virtually all aspects of RNA metabolism. It exhibits robust RNA helicase activity in vitro. However, little is known about the molecular and physical determinants for RHA substrate recognition and RHA translocation along the nucleic acids. Here, our non-denaturing polyacrylamide gel electrophoresis (PAGE)-based unwinding assays of chemical and structural modified substrates indicate that RHA translocates efficiently along the 3’ overhang of RNA, but not DNA, with a requirement of covalent continuity. Ribose-phosphate backbone lesions on both strands of the nucleic acids, especially on the 3’ overhang of the loading strand, affect RHA unwinding significantly. Furthermore, RHA requires RNA on the 3’ overhang which directly or indirectly connects with the duplex region to mediate productive unwinding. Collectively, these findings propose a basic mechanism of the substrates determinants for RHA backbone tracking during duplex unwinding.

KEYWORDS: RHA; Substrates continuity; Substrates specificity; Duplex junction; Unwinding mechanism


Page 2 of 25

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION RNA helicase A (RHA), also known as nuclear DNA helicase II (NDH II) and DHX9, is a member of DExH/D-box proteins and belongs to helicase superfamily II

1-3.

It was first purified

in 1991 from calf thymus nuclei as a DNA helicase 4, and subsequently characterized as the most abundant and stable RNA helicase in HeLa cells

5,6.

RHA unwinds duplexes by translocating

rapidly, processively and directionally from 3’ to 5’ with a defined minimal binding site size of 16 nucleotides 5,7. It participates in diverse cellular functions such as transcription 8-10, translation 11,12,

RNA interference

13

and innate immune response

14,15.

RHA can promote replication of a

number of viruses including hepatitis C virus (HCV) 16,17, foot-and-mouth disease virus (FMDV) 18,

influenza A virus 19 and human immunodeficiency virus 1 (HIV-1) 20-23. Besides, it also plays

a regulatory role in maintenance of genomic stability for unwinding intra-molecular triplex (HDNA), DNA- and RNA-containing forks, DNA- and RNA-containing displacement loops (Dand R-loop), and also G-quadruplexes 24-28. Although the multiple roles of RHA in cellular biology have been informative, its physical mechanism of nucleic acid recognition and translocation remains obscure. Recently, single molecule fluorescence resonance energy transfer technique has been used to investigate the molecular mechanism involved in RNA duplex unwinding by RHA, and showed the repetitive activity of RHA in the multi-step unwinding process 29. However, it remains unclear how RHA tracks along the 3’ loading strand (the strand bearing the 3’ overhang, also called bottom strand) of the duplex, and whether RHA can tolerate the perturbations to the covalent continuity (cleavage of phosphodiester bond 30) or to the nucleic acid continuity (e.g., oligo ethylene glycol linkers incorporated into the phosphodiester backbone

30)

in the substrates. Besides, previous

studies suggest that nucleotides specific SF2 helicases control substrate recognition through


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

specific interactions with the loading strand

31-33,

Page 4 of 25

it is of great interest to dissect the basis of

nucleotides specificity of RHA and to identify mechanistic differences in the unwinding mechanisms of RHA from other SF2 helicases 34-36. Here, we conducted unwinding analysis for a diverse family of modified duplex substrates to probe the unwinding mechanism of RHA and the respective role of each strand in the duplex. We found that RHA requires covalent continuity along the loading strand to mediate duplex unwinding, which is similar to another two members of DExH/D-box proteins: NPH-II and NS3. However, unlike NPH-II

30

or NS3

37

that can

translocate over major disruptions in the ribose-phosphate backbone either in the top strand (complementary to the bottom strand) or both strands respectively, RHA cannot tolerate nucleic acid discontinuities in the substrates, especially in the bottom strand. Besides, we observed that RNA in the single strand region which is immediately adjacent to the duplex region of the substrates is critical to the helicase activity of RHA. If DNA is placed adjacent to the duplex, productive unwinding by RHA can also be promoted by a single-stranded RNA tail linked after the DNA. Collectively, we propose a more accurate and complete model to reveal the unwinding process of RHA. MATERIAL AND METHODS Protein expression and purification Recombinant RHA protein was expressed using the Baculovirus expression system. The recombinant pFASTBac-RHA-HIS vector that contains full-length DHX9 gene sequence and a 6×His tag was transformed into MAX Efficiency DH10Bac cells for DHX9 bacmid formation. Baculovirus stock was generated by transfecting DHX9 bacmid into SF9 cells. 500 mL serumindependent suspension of SF9 cells with a cell density of 2.5×106 were infected with virus stock at an MOI of 1. Cells were harvested after 72 h infection and all purification steps were carried


Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

out at 4 C. After washing with ice-cold 1×PBS buffer, the whole-cell pellets were homogenized in ice-cold hypotonic lysis buffer (25 mM HEPES-KOH pH 7.9, 10 mM NaCl, 10 mM Na2S2O5, 4 mM MgCl2, 7 mM β-ME and 1×Protease Inhibitor) followed by addition of an equal volume of hypertonic buffer (25 mM HEPES-KOH pH 7.9, 1 M NaCl, 10 mM Na2S2O5, 4 mM MgCl2, 7 mM β-ME, 1.6 mM imidazole, 30% glycerol and 1×Protease Inhibitor). The lysate was incubated on ice for 30 min (several times of sonication were optional for abundant lysis) and then centrifuged for 20 min at 15000 rpm. Primary purification of the supernatant was performed by affinity chromatography after 2 h incubation with Ni2+-NTA-agarose (Invitrogen). Protein fractions were eluted by the elution buffer I (25 mM HEPES-KOH pH 7.9, 1 M NaCl, 10 mM Na2S2O5, 4 mM MgCl2, 7 mM β-ME, 15% glycerol and 1×Protease Inhibitor) that with a continuous gradient of imidazole. After diluting the NaCl concentration of the Ni2+-eluted fraction to 50 mM, ion-exchange chromatography was conducted with Capto DEAE sepharose (GE) for further purification. The fractions were eluted by the elution buffer II (25 mM HEPESKOH pH 7.9, 10% glycerol and 1×Protease Inhibitor) that with a continuous gradient of NaCl. SDS-PAGE and Coomassie staining were performed to assess the protein purity (Figure S1 in the supplementary information). Finally, the NaCl concentration of the purified fractions was decreased to 50 mM by ultrafiltration with 30 KDa microsep (Millipore). Protein concentration was determined using the Bradford Coomassie protein assay kit (Sigma). Pure RHA were placed at -80 C for long-term storage. Synthesis of nucleic acid substrates All oligonucleotides (DNA oligonucleotides, RNA oligonucleotides, RNA oligonucleotides containing an 18-atom spacer polyglycol linker and RNA chimeric RNA-DNA oligonucleotides) were purchased from TAKARA. Substrate duplexes were prepared with a bottom strand that has


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a 3’-overhang and a top strand that is 3’-end labelled with FAM. The single-stranded RNAs without fluorescein labelling which were identical to the sequence of the top strands were used as trap RNAs, unless stated otherwise. Sequences of the above substrates were given in the Table S1 in the supplementary information. All oligonucleotides concentrations were determined by Nanodrop. RHA unwinding assays All unwinding assays were performed using standard reaction conditions which were established in our laboratory (20 mM Tris-HCl pH 7.4, 20 mM NaCl, 3 mM MgCl2, 0.1 mg/mL BSA, 2 mM DTT, 1 unit/μL RNasin, 40 nM RHA protein, 19 C), unless stated otherwise. Prior to initiation of unwinding reactions, RHA protein was pre-incubated with duplex substrate (4 nM) for 5 min at 19 C to promote formation of a functional complex. Unwinding reactions were initiated by adding 3 mM ATP and 5 nM unlabeled ssRNA (trap RNA). The excess trap RNA with identical sequence to the labeled top strand was used to prevent reannealing of the unwound strand (Figure S2 in the supplementary information). All reactions were conducted in volumes of 20 μL at 19 C. Specified time points were taken by adding equal volume of stop buffer (50 mM EDTA, 1% SDS, 20% glycerol). Duplex and unwound products were resolved by 12% (w/v) native PAGE. Size markers for unwound species were made by heating duplex substrates at 95 C for 5 min. The gels were visualized on Typhoon and the reaction products were analyzed using ImageJ software. The proportions of the unwound fractions at each time points were calculated by the formula of ft,unwound/(ft,unwound+ft)- f0/(f0,unpaired+f0) (f0 and ft represented the gray value of the double-stranded substrates at initial time point and the following time point t respectively; f0,unpaired and ft,unwound represented the corresponding gray value of the singlestranded substrates at initial time point and the following time point t respectively). Kinetic


Page 6 of 25

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

analysis of various substrate duplexes was implemented using KaleidaGraph software, and data points were fitted to the integrated first-order rate law. Error analysis was conducted by using at least three trials for each experiment. RESULTS RHA requires covalent continuity along the loading strand To investigate the substrate features important for unwinding by RHA, a series of RNA substrates that contain structural and chemical modifications were synthesized in either the top or the bottom strands, and their sequences were given in the Table S1 in the supplementary information. First, we examined the effects of disruptions in the backbone continuity through designing RNA substrates containing nicks. These substrates were created by annealing one continuous strand of RNA to a set of two adjacent pieces of complementary RNAs. As shown in Figure 1, the substrate RNAa which has no nicks was partially unwound by RHA (Figure 1A). Introducing a top-strand nick into this substrate to form RNAb increased the amounts of unwound fractions for both labelled top-strand fragments which were almost all released (Figure 1B, species i and ii). However, for the substrate RNAc bearing a bottom-strand nick, RHA unwinding stopped at the position of the nick only releasing the labelled bottomstrand preceding the nick (Figure 1C, species iii). These data indicate that the two strands of the substrate are differentiated by RHA during unwinding, and that the physical continuity of the bottom strand is of primary importance for RNA recognition and translocation. The fact that the top-nicked substrate showing more unwinding than no-nicked RNAs is also consistent with reported results that RHA prefers substrates with longer 3’-overhang and shorter duplex top-nicked substrate exhibited longer 3’ overhang after releasing species i).


5

(the

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Strand continuity requirement of RHA. The two RNAs used to make the nicked strand were each 3’-end labelled with FAM, as indicated by asterisk. For each panel, the first lane indicates the extent of unwinding after 30 min at 37 C. The fourth lane was loaded with a 95 C heat control to indicate the gel running position of the two labelled strands flanking the nick. (A) RHA unwinding of substrate RNAa. Trap A was used as trap RNA. (B) RHA unwinding of substrate RNAb with a nick in the top strand. Trap A was used as trap RNA. (C) RHA unwinding of substrate RNAc with a nick in the bottom strand. Trap B was used as trap RNA. RHA unwinding with substrates bearing disruptions of nucleic acid continuity Based on the above results that RHA requires physical continuity along the bottom strand to mediate duplex unwinding, we further investigated whether RHA unwinding simply requires covalent continuity, or a continuous strand of the nucleic acids. To approach this question, we synthesized a family of substrates in which 18-atom polyglycol linkers spanning three bases were incorporated into the phosphodiester backbone of either strand (substrates S1-S4 and L7L8, Figure 2 and Table S1 in the supplementary information). Unlike the nicks of the substrates,


Page 8 of 25

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

this chemical modification disrupted the nucleic acid continuity without physically breaking the covalent continuity of the backbone. First we tested RNA substrates containing a 16 nt 3’ overhang and a 19 bp duplex (Figure 2A). To explore positional effects of the polyglycol linkers, we placed the linker at sites closer to (substrates S1 and L1, Figure 2A) or farther from (substrates S2 and L2, Figure 2A) the junction which indicates the joint of the single-stranded region and the paired region. The unwinding amplitudes of substrates S1 and S2 bearing a top-strand linker were both reduced by half compared to that of substrate RNA1 (without linker) regardless of the linker position. However, for substrates L1 and L2, which contain a bottom-strand linker, the linker position made a remarkable difference at the unwinding efficiency of RHA. The declining unwinding amplitude of substrate L1 compared with substrate RNA1 was nearly three times down than that of substrate L2. According to previous studies that RHA unwinding efficiency increases with increasing 3’end overhang length 5, we further used RNA substrates with a longer single-stranded tail of 24 nt to examine whether the unwinding inhibition caused from the linker could be compromised by longer overhang. As shown in Figure 2B, inserting linkers also caused decrease in unwinding efficiency of each substrate (substrates S3, S4, L3 and L4), especially the one that contains linkers adjacent to the junction at the loading strand (substrates L3). Besides, compared to the substrates RNA1, S1, S2, L1 and L2, respectively, the unwinding amplitudes of substrates RNA2, S3, S4, L3 and L4 all showed about 5% increase. These results together indicate that except for an essential requirement of covalent continuity along the bottom strand (loading strand), nucleic acid continuity of both strands are also important for RHA unwinding, especially


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in the position adjacent to the junction at the loading strand, and increasing tail length cannot offset the effects from nucleic acid discontinuity. Furthermore, given that substrates L1 and L3, substrates L2 and L4 had 5 bp and 12 bp between the junction and the linker, respectively, we suspected that if the shorter distance to the junction was the reason that L1 and L3 showed lesser unwound, and if there was a critical binding site for RHA between 5 bp and 12 bp to the junction that might affect RHA unwinding efficiency directly. To this end, substrates L5 and L6 which respectively had 7 bp and 10 bp between the junction and the linker were designed. As shown in Figure 2C, even though the site of linker modification in substrate L6 was just next to that in substrate L5, the unwinding amplitude of substrate L5 significantly decreased four times than that of substrate L6. It appeared that a 10 nt binding site in the duplex region from the junction is critical for RHA unwinding. To further confirm whether unwinding efficiency is related to the distance from the linker to the junction, we created substrate RNA3 with longer duplex region (27 bp with a 16 nt overhang) and its bottom-linker modified versions (substrates L7 and L8). Like substrates L5 and L6, the site of the linker insertion in substrate L7 adjoin to that in substrate L8 having 10 bp and 13 bp to the junction, respectively. Unexpectedly, although there is a 10 nt binding site in the duplex region of L7 and L8, both substrates L7 and L8 showed a significant inhibition for RHA unwinding activity (Figure 2D). Therefore, we speculate that the higher unwinding amplitudes of substrates L2, L4 and L6 compared to substrates L1, L3, L5, L7 and L8 are irrelevant to the junction, but are attributed to their shorter distance (less than or equal to 6 bp) from the linker to the 5’-end of the loading strand. It is most likely that RHA can only unwind to the linker region which blocks RHA translocation, while the remaining base pairs following the linker have to


Page 10 of 25

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

dissociate spontaneously. L2, L4, L6 have fewer base pairs (4-6 bp), thus easier to separate spontaneously even at 19 C.

Figure 2. Nucleic acid continuity requirement of RHA. (A) Reaction time courses of substrates RNA1, S1, S2, L1 and L2 containing 16 nt 3’ overhang. Unwinding rate constants (k) and final reaction amplitudes (Af) were Af(RNA1) = 0.74 ± 0.02, k(RNA1) = 0.14 ± 0.01 min-1; Af(S1) = 0.30 ± 0.01, k(S1) = 0.60 ± 0.08 min-1; Af(S2) = 0.35 ± 0.01, k(S2) = 0.40 ± 0.06 min-1; Af(L1) = 0.05 ± 0.01, k(L1) = 0.04 ± 0.05 min-1; Af(L2) = 0.34 ± 0.01, k(L2) = 0.32 ± 0.05 min-1. (B) Reaction time courses of substrates RNA2, S3, S4, L3 and L4 containing 24 nt 3’ overhang. Af(RNA2) = 0.79 ± 0.03, k(RNA2) = 0.13 ± 0.01 min-1; Af(S3) = 0.35 ± 0.01, k(S3) = 0.42 ± 0.05 min-1; Af(S4) = 0.40 ± 0.01, k(S4) = 0.25 ± 0.03 min-1; Af(L3) = 0.12 ± 0.01, k(L3) = 0.03 ± 0.01 min-1; Af(L4) = 0.39 ± 0.01, k(L4) = 0.19 ± 0.03 min-1. (C) Reaction time courses of substrates L5 and L6 containing 24 nt 3’ overhang. Af(L5) = 0.10 ± 0.01, k(L5) = 0.04 ± 0.01 min-1; Af(L6) = 0.35 ± 0.02, k(L6) = 0.15 ± 0.02


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

min-1. (D) Reaction time courses of substrates RNA3, L7 and L8 containing 16 nt 3’ overhang. Af(RNA3) = 0.61± 0.01, k(RNA3) = 0.12 ± 0.02 min-1; Af(L7) = 0.08 ± 0.01, k(L7) = 0.02 ± 0.01 min-1; Af(L8) = 0.05 ± 0.01, k(L8) = 0.04 ± 0.01 min-1. Schemes of RNA substrates with polyglycol modifications are presented on the right of each figure. The black curves represent 18-atom polyglycol linkers that span three bases in the duplex region. The linker was placed in either the top (substrates S1, S2, L3 and L4) or the bottom strand (substrates L1, L2, L3, L4, L5, L6, L7 and L8). FAM were labelled at the 3’ terminal of the top strands and indicated as asterisks. RNA1, RNA2 and RNA3 were presented as controls indicating the unwinding activity of RHA for substrates without linkers. Trap C and Trap D were used as trap RNA for 19 bp and 27 bp duplex substrates respectively. All the reactions were performed at 19 C to monitor the unwinding reactions more accurately. RHA unwinding with DNA/RNA hybrid substrates and chimeric substrates In view of the important role that nucleotides specificity play in the interaction between helicase and their substrates, we created a pair of hybrid duplex substrates (substrates H1 and H2, Figure 3A) and a set of chimeric substrates (substrates H3-H7, Figure 3B) to dissect this property of RHA. These substrates were analogous to RNA2, and only certain portion of RNA2 was replaced by DNA with their sequences presented in Table S1 in the supplementary information (substrates H1-H7). As shown in Figure 3A, substrate H1, which contained a DNA bottom strand and an RNA top strand, was not unwound by RHA under our experimental conditions. In contrast, substrate H2, which contained an RNA bottom strand and a DNA top strand, was readily unwound by RHA. But the rate constant and amplitude for unwinding were respectively higher and lower than that of RNA2. Perhaps because RNA2 has higher affinity to RHA and it is also more stable than the


Page 12 of 25

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

hybridized duplexes. These results demonstrate that the top and bottom strands of the substrate are recognized differently by RHA, and also provide further evidence that RHA tracks along the loading strand. Having established that an all-DNA loading strand was inhibitory for RHA unwinding, we wondered whether this inhibition effect was caused by the duplex region or the overhang region. To examine this question, we created substrates H3 and H4, which contained DNA either throughout the duplex region or throughout the overhang of the loading strand, respectively. RHA was unable to unwind substrate H4 under our reaction conditions, while unwinding of substrate H3 was still significant (Figure 3B). These data indicate that RHA requires RNA overhang to mediate efficient duplex unwinding. Based on this, we tested which portion of the overhang contributed the most to helicase activity, by using a set of substrates with an RNA section placed at different positions along the overhang region (substrates H5, H6 and H7, Figure 3B). Given that the minimal binding site of RHA is 16 nucleotides which includes residues in the duplex region 7, we designed the RNA section with a length either longer or shorter than 16 nt. We observed that substrate H5 containing 4 nt of RNA in the overhang adjacent to the junction was unwound most efficiently by RHA compared to other DNA containing substrates at the overhang region (Figure 3B). Substrate H6 containing 20 nt of RNA in the overhang away from the junction, and substrate H7 containing 12 nt of RNA in the overhang away from the junction could also be unwound by RHA, but with a less extent than substrate H5, indicating that the position and the length of RNA component in the overhang are essential in efficient unwinding of RHA. Furthermore, we examined whether increasing enzyme concentration could affect the unwinding efficiency of H5, H6 and H7. We observed that substrates H5, H6 and H7 were more


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

unwound with increasing RHA concentration (Figure S3 in the supplementary information), suggesting that DNA components in the loading strand may affect the binding affinity of RHA.

Figure 3. Oligonucleotides composition requirement of RHA. (A) Reaction time courses of RNA2 and hybrid substrates. Af(RNA2) = 0.79 ± 0.03, k(RNA2) = 0.13 ± 0.01 min-1; Af(H2) = 0.67 ± 0.01, k(H2) = 0.91 ± 0.02 min-1. Trap C was used as trap RNA for RNA2 and H1, while Trap E was used as trap RNA for H2. (B) Reaction time courses of substrates with DNA in various parts of the loading strand. Af(H3) = 0.23 ± 0.01, k(H3) = 0.04 ± 0.01 min-1; Af(H5) = 0.25 ± 0.01, k(H5) = 0.13 ± 0.03 min-1; Af(H6) = 0.19 ± 0.01, k(H6) = 0.04 ± 0.003 min-1; Af(H7) = 0.18 ± 0.03, k(H7) = 0.01 ± 0.003 min-1. Trap C was used as trap RNA. Schemes of substrates used in this experiment were presented on the right of each figure, where DNA and RNA are coloured in red and black respectively. FAM was labelled at the 5’ terminal of the top strands and indicated as asterisks. RNA2 served here as control. All the reactions were performed at 19 C to monitor the unwinding reactions more accurately. DISCUSSION Physical continuity of the loading strand is essential for RHA unwinding


Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Here we show that RHA cannot proceed through physical breaks as it translocates along the loading strand during duplex unwinding, and nucleic acid discontinuity is inhibitory wherever in the top or the bottom strand of an RNA substrate. Given that RHA belongs to SF2 helicases, we compare the translocation strategies of SF2 helicase NPH-II and NS3 with RHA. We find that these three helicases all require covalent continuity on the loading strand. However, RHA differs from them in that linker modifications on the top strand can also inhibit RHA unwinding, while the effect is more moderate than that on the bottom strand. In contrast, NPH-II can unwind RNA duplexes containing polyglycol linkers in the top strand with negligible effects 30, while NS3 can tolerate the linkers either in the top strand or the bottom strand 37. The previous study attributed the robust translocation of NS3 to its longer kinetic step size when compared with NPH-II (6 bp for NPH-II while 14~18 bp for NS3) 37-39, but our unwinding analysis for RHA demonstrates that having relatively large binding site size (16 nucleotides for RHA)

7

does not really help in

translocating over polyglycol linkers. Thus, we suggest that RHA clamps onto the backbone of the loading strand and then transits along it, accompany with interaction with the top stand. RHA shows more sensibility to the subtle perturbations of the loading strand in contrast with that of the top strand, perhaps because the top strand backbone contacts only play an auxiliary role during translocation. Oligonucleotide specificity is mainly determined by the loading strand Based on the comparison between RNA duplexes, RNA with polyglycol linkers (substrates S1S4, L1-L8, Figure 2) and RNA-DNA hybrid duplexes (substrates H1 and H2, Figure 3), we demonstrate that the oligonucleotide specificity is dictated by the loading strand, and the nucleotides identity of the top strand has less effect on the primary function of RHA. According to the previous reports, RHA can only unwind substrates possessing 3’ single-stranded overhang


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

and its unwinding efficiency increases with the overhang length (substrates RNA1, RNA2 and RNA3, Figure 2). For most unidirectional helicase, this single-stranded overhang may make contributions in providing polarity and electrostatic determinants for productive binding

33.

Unwinding studies on substrates H3 (containing DNA throughout the duplex region of the loading strand) and H4 (all-DNA overhang) show that, different from NPH-II which has a requirement of RNA in the duplex region of the bottom loading strand 33, RHA requires RNA in the overhang to mediate duplex unwinding (Figure 3B). RHA unwinds a small amount of the substrate H5 (containing 4 nt of RNA in the overhang adjacent to the junction) but fails to unwind substrate H4 (all-DNA overhang) or substrate with an RNA overhang shorter than 5 nt, suggesting that the several nucleotides in the overhang which are immediately adjacent to the junction play a critical role in the recognition between RHA and substrate, and that the remaining portion of the overhang also functions in supporting RHA binding even if it is DNA. The unwinding efficiency improves with increasing length of the single-stranded RNA tail which is not directly physically linked to the duplex region, suggesting that RHA may utilize the RNA tail to initiate unwinding. Notably, there are also reports that RHA can unwind a small amount of hybrid substrate that takes DNA as the loading strand and RNA as the top strand

1,26.

Although we did not observe

unwinding of substrates H1 and H4 under our experimental conditions, we obtained the consistent results after increasing RNA trap and RHA concentration and reaction temperature to the concentrations reported in the literature 26 (Figure S4 in the supplementary information), and we observed a small portion of substrates H1 and H4 was unwound. It is most likely that more RNA trap may provide more potent inhibition in reannealing of the duplex regions that are flanking and captured by RHA occasionally, especially at higher temperature. To monitor the


Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

unwinding reactions more accurately, we chose our unwinding condition at 19 C, so that the unwinding speed is not too fast to monitor and duplex flanking is not too significant as well. Mechanism and significance for substrates unwinding by RHA Based on the data presented here, we propose a mechanism to explain the unwinding process of RHA (Figure 4). We divide this mechanism into two phases: loading phase and translocating phase. In loading phase, RHA initiates unwinding from loading onto the junction of the duplex from the 3’ overhang in an ATP-dependent fashion. Given that RHA binds RNA with higher affinity than DNA 5, RHA shows more stable binding with substrate when all or critical portion of the loading strand is RNA. If DNA component is placed adjacent to the duplex region, productive loading of RHA onto the substrate can also be facilitated by the single-stranded RNA tail connected after the DNA. In the translocating phase, RHA shows more efficient movement on RNA than DNA due to its nucleic acid specificity. The lesion placed at the loading strand hinders RHA translocation, so RHA may dissociate from the loading strand, or return to the unwinding initial site due to its repetitive unwinding characteristic 29. By contrast, lesion in the top strand does not play a critical role in the progress of RHA, even though it partly inhibits RHA translocation. Since RHA can unwind substrates containing lesions that can be found in natural RNAs forming coaxial staking, it may be possible that RHA is involved in remodeling RNA structures containing coaxial staking interactions 40, or RNA structures containing coaxial staking interactions may regulate RHA activities. In fact, RHA has been shown to remodel DNA triplexes containing coaxial staking 25. This mechanism is partially consistent with the reported one strand-exclusion model which is widely used in explaining translocation of processive helicases 41,42. These results have enriched the understanding of substrates determinants for RHA


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

unwinding. The proposed model here may also help to investigate the biological functions of other helicases.

Figure 4. The mechanism of duplex unwinding by RHA. RNA, DNA and RHA are coloured in grey, pink and yellow, respectively. The solid arrow represents major pathway and the dash arrow represents minor pathway with wider arrow indicating higher rate. In the major pathway, RHA first tightly binds to the RNA at the junction, and then efficiently translocates along the 3’ overhang of the loading strand of the RNA, removing the top strand during translocation; In the minor pathway, RHA may load onto the RNA component placed away from the junction or directly bind to the DNA with low stability, and then inefficiently translocates along the DNA until getting to the junction and initiate unwinding of the RNA duplex. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Funding Sources This work was supported by the National Natural Science Foundation [31522056 to F.L.]; and the National Key Research and Development Program [2015BAD12B01, 2018YFD0500100 to F.L.].


Page 18 of 25

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Note The authors have no conflicts of interest on this work. SUPPORTING INFORMATION Table S1. Sequences of substrates used in this work. Figure S1. Purification of RHA. Figure S2. Trap RNA titration. Figure S3. Reaction time courses of duplex unwinding of substrates H5, H6 and H7 with 80 nM RHA and 3 mM ATP/Mg2+ at 19 C. Figure S4. Duplex unwinding of substrate H1 and H4 by RHA at 37 C with higher trap RNA concentration. REFERENCES (1) Zhang, S. and Grosse, F. (2010) Molecular characterization of nuclear DNA helicase II (RNA helicase A). Methods Mol. Biol., 587, 291-302. (2) Xing, L., Niu, M., Zhao, X. and Kleiman, L. (2014) Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell. Biochim. Biophys. Acta., 1840, 2234-2243. (3) Fidaleo, M., De Paola, E. and Paronetto, M.P. (2016) The RNA helicase A in malignant transformation. Oncotarget, 7, 28711-28723. (4) Zhang, S.S. and Grosse, F. (1991) Purification and characterization of two DNA helicases from calf thymus nuclei. J. Biol. Chem., 266, 20483-20490. (5) Lee, C.G. and Hurwitz, J. (1992) A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J. Biol. Chem., 267, 4398-4407.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(6) Lee, C.G. and Hurwitz, J. (1993) Human RNA helicase A is homologous to the maleless protein of Drosophila. J. Biol. Chem., 268, 16822-16830. (7) Zhang, S. and Grosse, F. (1994) Nuclear DNA helicase II unwinds both DNA and RNA. Biochemistry, 33, 3906-3912. (8) Zhang, S. and Grosse, F. (1997) Domain structure of human nuclear DNA helicase II (RNA helicase A). J. Biol. Chem., 272, 11487-11494. (9) Fuller-Pace, F.V. (2006) DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation. Nucleic Acids Res., 34, 4206-4215. (10) Cordin, O., Hahn, D. and Beggs, J.D. (2012) Structure, function and regulation of spliceosomal RNA helicases. Curr. Opin. Cell Biol., 24, 431-438. (11) Hartman, T.R., Qian, S., Bolinger, C., Fernandez, S., Schoenberg, D.R. and Boris-Lawrie, K. (2006) RNA helicase A is necessary for translation of selected messenger RNAs. Nat. Struct. Mol. Biol., 13, 509-516. (12) Chen, C.Y., Liu, X., Boris-Lawrie, K., Sharma, A. and Jeang, K.T. (2013) Cellular RNA helicases and HIV-1: insights from genome-wide, proteomic, and molecular studies. Virus Res., 171, 357-365. (13) Robb, G.B. and Rana, T.M. (2007) RNA helicase A interacts with RISC in human cells and functions in RISC loading. Mol. Cell., 26, 523-537. (14) Zhang, Z., Yuan, B., Lu, N., Facchinetti, V. and Liu, Y.J. (2011) DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells. J. Immunol., 187, 4501-4508. (15) Lee, T. and Pelletier, J. (2016) The biology of DHX9 and its potential as a therapeutic target. Oncotarget, 7, 42716-42739.


Page 20 of 25

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(16) Isken, O., Baroth, M., Grassmann, C.W., Weinlich, S., Ostareck, D.H., Ostareck-Lederer, A. and Behrens, S.E. (2007) Nuclear factors are involved in hepatitis C virus RNA replication. RNA, 13, 1675-1692. (17) He, Q.S., Tang, H., Zhang, J., Truong, K., Wong-Staal, F. and Zhou, D. (2008) Comparisons of RNAi approaches for validation of human RNA helicase A as an essential factor in hepatitis C virus replication. J. Virol. Methods, 154, 216-219. (18) Lawrence, P. and Rieder, E. (2009) Identification of RNA helicase A as a new host factor in the replication cycle of foot-and-mouth disease virus. J. Virol., 83, 11356-11366. (19) Lin, L., Li, Y., Pyo, H.M., Lu, X., Raman, S.N., Liu, Q., Brown, E.G. and Zhou, Y. (2012) Identification of RNA helicase A as a cellular factor that interacts with influenza A virus NS1 protein and its role in the virus life cycle. J. Virol., 86, 1942-1954. (20) Li, J., Tang, H., Mullen, T.M., Westberg, C., Reddy, T.R., Rose, D.W. and Wong-Staal, F. (1999) A role for RNA helicase A in post-transcriptional regulation of HIV type 1. Proc. Natl. Acad. Sci. U. S. A., 96, 709-714. (21) Fujii, R., Okamoto, M., Aratani, S., Oishi, T., Ohshima, T., Taira, K., Baba, M., Fukamizu, A. and Nakajima, T. (2001) A Role of RNA Helicase A in cis-Acting Transactivation Response Element-mediated Transcriptional Regulation of Human Immunodeficiency Virus Type 1. J. Biol. Chem., 276, 5445-5451. (22) Bolinger, C., Sharma, A., Singh, D., Yu, L. and Boris-Lawrie, K. (2010) RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions. Nucleic Acids Res., 38, 16861696. (23) Xing, L., Liang, C. and Kleiman, L. (2011) Coordinate roles of Gag and RNA helicase A in promoting the annealing of formula to HIV-1 RNA. J. Virol., 85, 1847-1860.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(24) Qin, S.L., Xie, A.G., Bonato, M.C. and McLaughlin, C.S. (1990) Sequence analysis of the translational elongation factor 3 from Saccharomyces cerevisiae. J. Biol. Chem., 265, 1903-1912. (25) Jain, A., Bacolla, A., Chakraborty, P., Grosse, F. and Vasquez, K.M. (2010) Human DHX9 helicase unwinds triple-helical DNA structures. Biochemistry, 49, 6992-6999. (26) Chakraborty, P. and Grosse, F. (2010) WRN helicase unwinds Okazaki fragment-like hybrids in a reaction stimulated by the human DHX9 helicase. Nucleic Acids Res., 38, 47224730. (27) Chakraborty, P. and Grosse, F. (2011) Human DHX9 helicase preferentially unwinds RNAcontaining displacement loops (R-loops) and G-quadruplexes. DNA Repair, 10, 654-665. (28) Jain, A., Bacolla, A., Del Mundo, I.M., Zhao, J., Wang, G. and Vasquez, K.M. (2013) DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells. Nucleic Acids Res., 41, 10345-10357. (29) Koh, H.R., Xing, L., Kleiman, L. and Myong, S. (2014) Repetitive RNA unwinding by RNA helicase A facilitates RNA annealing. Nucleic Acids Res., 42, 8556-8564. (30) Kawaoka, J., Jankowsky, E. and Pyle, A.M. (2004) Backbone tracking by the SF2 helicase NPH-II. Nat. Struct. Mol. Biol., 11, 526-530. (31) Shuman, S. (1993) Vaccinia virus RNA helicase. Directionality and substrate specificity. J. Biol. Chem., 268, 11798-11802. (32) Lohman, T.M. and Bjornson, K.P. (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem., 65, 169-214. (33) Kawaoka, J. and Pyle, A.M. (2005) Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II. Nucleic Acids Res., 33, 644-649.


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(34) Gao, Z., Putnam, A.A., Bowers, H.A., Guenther, U.P., Ye, X., Kindsfather, A., Hilliker, A.K. and Jankowsky, E. (2016) Coupling between the DEAD-box RNA helicases Ded1p and eIF4A. Elife, 5, e16408. (35) Pugh, R.A., Honda, M., Leesley, H., Thomas, A., Lin, Y., Nilges, M.J., Cann, I.K. and Spies, M. (2008) The iron-containing domain is essential in Rad3 helicases for coupling of ATP hydrolysis to DNA translocation and for targeting the helicase to the single-stranded DNAdouble-stranded DNA junction. J. Biol. Chem., 283, 1732-1743. (36) Gilman, B., Tijerina, P. and Russell, R. (2017) Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs. Biochem. Soc. Trans., 45, 1313-1321. (37) Beran, R.K., Bruno, M.M., Bowers, H.A., Jankowsky, E. and Pyle, A.M. (2006) Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. J. Mol. Biol., 358, 974-982. (38) Jankowsky, E., Gross, C.H., Shuman, S. and Pyle, A.M. (2000) The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature, 403, 447-451. (39) Serebrov, V. and Pyle, A.M. (2004) Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature, 430, 476-480. (40) Devi, G., Zhou, Y., Zhong, Z., Toh, K.D. and Chen, G. (2015) RNA triplexes: from structural principles to biological and biotech applications. Wiley Interdiscip. Rev. RNA, 6, 11128. (41) Gai, D., Chang, Y.P. and Chen, X.S. (2010) Origin DNA melting and unwinding in DNA replication. Curr. Opin. Struct. Biol., 20, 756-762.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42) Syed, S., Pandey, M., Patel, S.S. and Ha, T. (2014) Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase. Cell Rep., 6, 1037-1045.


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

For table of contents use only 82x32mm (300 x 300 DPI)


D-box

Recommend Documents