Studying structure and function of spliceosomal helicases

Methods 125 (2017) 63–69

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Methods journal homepage: www.elsevier.com/locate/ymeth

Studying structure and function of spliceosomal helicases Ralf Ficner ⇑, Achim Dickmanns, Piotr Neumann Department of Molecular Structural Biology, Institute of Microbiology and Genetics, GZMB, Georg-August-University Göttingen, 37077 Göttingen, Germany

a r t i c l e

i n f o

Article history: Received 10 March 2017 Received in revised form 21 June 2017 Accepted 24 June 2017 Available online 29 June 2017

a b s t r a c t The splicing of eukaryotic precursor mRNAs requires the activity of at least three DEAD-box helicases, one Ski2-like helicase and four DEAH-box helicases. High resolution structures for five of these spliceosomal helicases were obtained by means of X-ray crystallography. Additional low resolution structural information could be derived from single particle cryo electron microscopy and small angle X-ray scattering. The functional characterization includes biochemical methods to measure the ATPase and helicase activities. This review gives an overview on the techniques used to gain insights in to the structure and function of spliceosomal helicases. Ó 2017 Elsevier Inc. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. X-ray crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Small angle X-ray scattering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Single particle electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. ATPase activity assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Helicase activity assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. RNA binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The splicing of pre-mRNA occurs in the nucleus of eukaryotic cells and is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein complex. For each intron to be removed from a pre-mRNA the spliceosome has to assemble newly in a stepwise and well-ordered fashion (Fig. 1). This requires the sequential assembly of five so called snRNPs (small nuclear RiboNucleoproteinParticles) termed U1, U2, U5, U4/U6, and a multitude of additional proteins like SF1/BBP and U2AF (for review see: [1]). Briefly, the U1 snRNP binds to the 50 splice site, while the SF1/ BBP and U2AF proteins bind to the branch point site (BPS) and the adjacent polypyrimidine tract yielding the spliceosomal E complex. In an ATP-dependent step the U2 snRNP joins and binds to ⇑ Corresponding author. E-mail address: [email protected] (R. Ficner). http://dx.doi.org/10.1016/j.ymeth.2017.06.028 1046-2023/Ó 2017 Elsevier Inc. All rights reserved.

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BPS leading to the displacement of SSF1/BBP and the formation of the A complex. Subsequently the U4/U6.U5 tri-snRNP joins the complex forming the B complex, which is still catalytically inactive. Large conformational and compositional rearrangements lead to the release of U1 and U4 snRNPs resulting in the Bact complex which is subsequently transformed into the activated B⁄ spliceosome. This B⁄ spliceosome catalyzes the first step of the splicing reaction resulting in the C complex. Additional rearrangements of the spliceosome lead to the C⁄ complex, which is competent to catalyze the second step of the splice reaction. The post-splicing complex is finally disassembled and the reaction products (mRNA and intron-lariat) are released. Almost all steps during the assembly of the spliceosome, the activation of the spliceosome, the splicing reactions itself, and the disassembly of the spliceosome are mediated by at least eight different ATP-dependent DExD/H-box helicases (Fig. 1) (for review see: [2,3]). Major model organisms for studying the role of these

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Fig. 1. Role of DExD/H-box proteins in pre-mRNA splicing. Simplified scheme of pre-mRNA splicing showing only pre-mRNA, UsnRNPs and splicoeosmal helicases. The DEADbox helicases Prp5, Sub2/UAP56 and Prp28 are required for the assembly of the spliceosome, the Ski2-like helicases Brr2 mediates a major rearrangement leading to the formation of the Bact spliceosome and the release of U1 and U4 snRNPs. The DEAH-box helicases Prp2, Prp16, Prp22 and Prp43 are necessary for the transition into a catalytically active spliceosome, the splicing process itself, the release of the spliced mRNA, and the disassembly of the post-catalytic spliceosome.

helicases in pre-mRNA splicing have been S. cerevisiae (sc), S. pombe (sp), H. sapiens (hs), and more recently for structural studies also C. thermophilum (ct). Many spliceosomal helicases were initially identified using yeast mutant strains. A large set of temperature-sensitive mutants, which were defective in pre-RNA processing and therefore denoted as prp mutants, have been described [4–6]. Several of these Prp proteins were found to be involved in pre-mRNA splicing and, after sequencing the corresponding genes, some Prp proteins could be assigned as members of the DExD/H-box protein family. By this approach the DExD/H-box proteins scPrp2 [7,8], scPrp5 [9], scPrp16 [10], scPrp22 [11], scPrp28 [12], scPrp43 [13] were found. Furthermore, the scBrr2 protein, which was identified by one of the cold-sensitive brr mutants of yeast, turned out to be another spliceosomal helicase that belongs to the subfamily of to Ski2-like helicases [14]. In case of Sub2/UAP56 helicase first the human UAP56 was found to be a spliceosomal DEAD-box protein [15], and later the S. cerevisiae and S. pombe orthologs were identified based on their sequence homology [16,17]. The human orthologs of the yeast spliceosomal helicases were found by the sequence analysis of the proteins present in purified snRNPs and/or spliceosomal complexes. By this approach, human spliceosomal proteins like U5 200K and U5 100K turned out to be the orthologs of Brr2 and Prp28, respectively [18–20]. The spliceosomal DExD/H-box proteins belong to the helicase superfamily 2 (SF2), which consists of a large number of monomeric RNA helicases involved in many processes such as translation initiation and termination, pre-mRNA editing and splicing, mRNA export as well as ribosome biogenesis. Based on their amino acid sequence the eight spliceosomal helicases are classified as either DEAD-box, DEAH-box or Ski2-like helicase. These families

not only differ in the amino acids of the name-giving DExD/H motif, but also in the their acceptance of exclusively ATP or any NTP, which is caused by absence or presence of another conserved sequence motif, the Q-motif (for review: [3]). Prp5, Sub2/UAP56 and Prp28 belong to the DEAD-box helicases, Brr2 is the only Ski2-like helicase, and Prp2, Prp16, Prp22 and Prp43 are DEAHbox helicases. Each of these spliceosomal helicases acts at a distinct step of the splicing process (Fig. 1). Prp5 and Sub2/UAP56 are required for formation of the pre-spliceosome [9,15,16,21]. Prp28 mediates the exchange of U1 with U6 at the 50 splice site [22]. Brr2 unwinds the U4/U6 snRNA duplex [23], which leads to the release of the U4 snRNP and the formation of the Bact complex (see review: [24]). Prp2 activates the spliceosome prior to the first esterification step [25] and drives the transition from the Bact to B⁄ complex [26]. Prp16 is required for the second catalytic step of the splicing reaction [27]. Prp22 is needed for release of spliced mRNA [11,28]. Prp43 is involved in the disassembly of the post-catalytic spliceosome [13] and was just recently shown to displace the U2 snRNP from post-catalytic spliceosomes [29]. Several of the spliceosomal helicases were reported to contribute to the fidelity of the splicing process by a kinetic proofreading mechanism (see review [30]. Such a proof-reading function was reported for Prp5 [31], Prp28 [22,32], Prp2 [33], Prp16 [34], and Prp22 [35]. Furthermore, Prp16 and Prp22 play an important role in the activation of alternative splice sites [36]. Besides the canonical spliceosomal DExD/H-box proteins that are conserved from yeast to human, the human multifunctional Upf1-like helicase Aquarius was shown to be part of a pentameric intron-binding complex and to be required for efficient pre-mRNA splicing [37]. In contrast to the spliceosomal DExD/H-box proteins, which all belong to the SF2, Aquarius is a member of the helicase superfamily 1 (SF1).

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The spliceosomal helicases have been investigated by different methods that will be summarized by this review.

2. Methods 2.1. X-ray crystallography Almost 90% of all known three-dimensional structures of biological macromolecules in the Protein Data Bank (PDB) were determined by means of X-ray crystallography (113,741 out of 127,184 deposited structures till end of Feb. 2017). The theoretical basis of macromolecular X-ray crystallography has been well described in several textbooks, e.g. [38,39]. Initiated by various structural genomics projects X-ray crystallography has become a semi-automated method, which takes advantage of highthroughput crystallization screening facilities, semi-automated synchrotron beam-lines, and robust routines for solving the crystallographic phase problems including the possibility of automated model building. Although structure refinement and map interpretation procedures became very powerful at moderate and high resolutions, structure determination and model building at lower than 3.5 Å resolution is still a challenging task. Recently, structure modeling by the program Rosetta was combined with the state-ofthe-art crystallographic software package Phenix, resulting in improved refinement and structure solution protocols at low resolution [40]. Despite the possibility to obtain structural models of good quality from diffraction data better than 5 Å resolution, the major bottleneck of the crystallographic structure determination is still the availability of well-diffracting single crystals. Especially crystals of proteins or protein-RNA complexes with a high intrinsic flexibility frequently diffract X-rays only poorly.

Hence, extensive experiments are required to optimize the crystallization conditions, or even to vary and optimize the used proteins and RNAs, e.g. by truncation [41,42]. Another approach could be the use of orthologous proteins from different organisms [43], which had led to a continuously increasing number of crystal structures of proteins (including spliceosomal helicases) from the thermophilic fungus Chaetomium thermophilum. For five out of the eight canonical spliceosomal DExD/H proteins high-resolution crystal structures could be determined (see Table 1), but high-resolution structures of Prp2, Pr16 and Prp22 are still missing. For Prp22, only the crystal structure of its C-terminal domains, and the NMR structure of its N-terminal S1 domain are available. For some of these spliceosomal helicases the crystallographic phase problem had to be solved de novo using a heavy atom derivative or selenomethionine-labeled protein crystals, while several other crystal structures could be solved by molecular replacement (for details see Table 1). Importantly, only for two spliceosomal helicases, namely Prp43 and Sub2, crystal structures with bound RNA are known. The Sub2RNA complex additionally contains a fragment of the nuclear export factor YRA1, since Sub2 is also involved in mRNA export. Furthermore, a low- resolution crystal structure of Sub2 in a complex with the export factors THO and Yra1 was also recently reported (Table 1).

2.2. Small angle X-ray scattering Small angle X-ray scattering allows to gain some structural information of biological macromolecules in solution. The theoretical basis for the application of SAXS on protein-RNA complexes was recently reviewed and the methods described in detail

Table 1 Crystal structures of spliceosomal helicases. HELICASE

Ligand

Organism

Method

PDB code

Resolution (Å)

References

Prp5 Prp5 UAP56 UAP56 UAP56-Mutant UAP56-RecA2 UAP56-RecA1 Sub2-YRA1c Sub2-THO-Yra1 Prp28 Prp28 Prp28 Brr2-Sec63 Brr2-Sec63 Brr2-Sec63 Brr2- Core Brr2-Core Mutant Brr2-Core Mutant Brr2-Jab1 complex Brr2-Jab1 Brr2_PWI Brr2-Jab1 Brr2-Jab1 Brr2-Jab1 Brr2-Jab1 Prp22_CTD Prp22_S1 Prp43 Prp43 Prp43 Prp43 Prp43 Prp43 Prp43 Aquarius

ADP – – ADP – – – RNA/ADP-BeF3

S. c. S. c. H. s. H. s. H. s. H. s. H. s. S. c. S. c. H. s. S. c. C. t. S. c. S. c. S. c. H. s. H. s. H. s. S. c. H. s. C. t. S. c. S. c. S. c. C. t. H. s. H. s. S. c. S. c. C. t. C. t. C. t. C. t. S. c. H. s.

SAD MR MR MR MR MAD MAD MR SAD SAD SAD MR MAD SAD MR MR MR MR SIRAS MR SAD MR MR MR MR MAD NMR SAD SAD MR MR MR MR MR SAD

4ljy 4lk2 1xti 1xtj 1xtk 1t5i 1t6n 5sup 5suq 4nho 4w7s 5dtu 3hib 3im1 3im2 4f91 4f92 4f93 4bgd 4kit 4rvq 5dca 5m52 5m5p 5m59 3i4u 2eqs 3kx2 2xau 5d0u 5lta 5ltj 5ltk 5jpt 4pj3

1.95 2.12 1.95 2.7 2.4 1.9 1.94 2.6 6.0 2.0 2.54 3.2 2.0 1.65 1.99 2.7 2.66 2.93 3.1 3.6 1.13 2.8 3.4 4.2 3.2 2.1

[72] [72] [61] [61] [61] [73] [73] [74] [74] [63] [58] [75] [76] Unpublished Unpublished [77] [77] [77] [78] [79] [80] [81] [82] [82] [82] [83] Unpublished [84] [85] [86] [68] [68] [68] [87] [37]

SO4 AMPPNP ADP – – – – – ATP/ADP ADP ADP – – – – – – ADP ADP ADP RNA/ADP-BeF3 ADP-BeF3 ADP-BeF3 CDP AMPPNP

2.2 1.9 2.9 2.6 1.8 3.2 2.93 2.3

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[44–46]. It is important to note, that SAXS provides only lowresolution information, and has its limitations for the de novo structure determination. However, SAXS is suitable for studying large conformational changes of proteins, for which high resolution crystal or NMR structures are already available, or to determine the global shape of macromolecular complexes, for which structures of the individual components are known. There has been only one report of a SAXS study on spliceosomal helicases, concerning Prp43 and the complex of Prp43 with the G-patch protein Pfa1 [44]. 2.3. Single particle electron microscopy Several recent reviews have described the method of single particle cryo electron microscopy (EM) and its current state of the art [47–49]. Various technological developments have dramatically improved cryo EM leading to EM maps interpretable with atomic models. This improvement was denoted as ‘‘resolution revolution”, as EM maps with an estimated resolution better than 3.5 Å could be obtained [50]. However, it appears misleading to designate the resulting EM structures with lower than 4 Å resolution as structures with ‘‘near-atomic resolution”, as atomic resolution corresponds to a resolution of 0.8 Å or better. Therefore, even a resolution of 2.0 Å is by far not ‘‘near-atomic”. Cryo-EM has recently provided structural models of the U4/U6. U5 tri-snRNP and of the spliceosome complexes Bact, C, C⁄, as well as the post-catalytic intron-lariat-spliceosome (ILS) complex. Some of the spliceosomal helicases are part of these complexes and therefore included in the cryo-EM structures (see Table 2). Brr2 is a central integral component of the U5 snRNP and therefor found in the EM structures of the tri-snRNP, the Bact and C complexes. Since high-resolution crystal structures of Brr2 had already be known, these could be used for the interpretation of the EM maps by fitting the high-resolution structure into the EM maps. The structures of the human and yeast Bact complex contain Prp2, for which no crystal structure has been available. Due to the high degree of similarity to Prp43, a homology model of Prp2 was generated based on the Prp43 crystal structure and fitted to the EM maps. However, these Prp2 models are still lacking the N-terminal 240 residues, as they are not conserved between

Prp43 and Prp2. Furthermore, the resolution of the part of the EM map covering Prp2 is much lower than the estimated overall resolution, which therefore hampers detailed structural analysis. For example, the resolution of the yeast Bact is reported to be 3.5 Å, while the local resolution for Prp2 appears to be 7.5 Å as estimated by Fourier Shell Correlation (FSC) calculated between the corresponding fragment of the 3D reconstruction and the model map. This reflects a general property of all spliceosome EM structures. The central and more rigid part of the complexes exhibits higher resolution, while the proteins on the periphery are much less resolved due to conformational and/or compositional heterogeneity. 2.4. ATPase activity assays Different assays were applied to measure the ATPase activity of spliceosomal DExD/H-box helicases. Some assays use radiolabeled ATP and separation of ATP, ADP and inorganic phosphate (Pi) by thin-layer chromatography (TLC), or alternatively use activated charcoal to separate [c-32P]ATP from the unbound 32Pi. Experimental details of these methods were reviewed recently [51,52]. A widely used non-radioactive method monitors the formation of Pi upon ATP hydrolysis by the reaction of Pi with molybdate, also known as malachite green assay [53,54]. A preferred method for kinetics measurements is a colorimetric coupled enzymatic assay using pyruvate kinase and lactate dehydrogenase, in which the oxidation of NADH+ is continuously monitored by measuring the absorbance at 340 nm [55]. A general feature of DExD/H-box proteins is the stimulation of their ATPase activity by RNA. Such RNA-dependent ATPase activity was overserved in vitro for scPrp2 [56], scPrp5 [57], scPrp16 [27], scPrp22 [11], scPrp28 [58], scBrr2 [59], scPrp43 [60], hsUAP56 [61], scSub2 [62]. However, an exception from that rule appears to be hsPrp28, which does not show ATPase activity, even in the presence of RNA [63]. Notably, hsPrp28 binds ATP only within the spliceosome, but not as purified protein [63]. Notably, advanced studies on the kinetics and thermodynamics of spliceosomal helicases by means of stopped-flow or quenchflow methods (for review see: [55]) have yet not been performed.

Table 2 Spliceosomal helicases in cryo-EM structures. Helicase complex

Organism

EMDB code

Resolution (Å)

PDB code

Helicase coordinates

Local resolution (Å)

Average RSCC

References

Prp2 in Bact

S.c.

EMD-4099

5.8

5lqw

9.9

0.43

[88]

Prp2 in Bact

S.c.

EMD-9524

3.5

5gm6

7.5

0.23

[89]

Prp16 in C

S.c.

EMD-4057

10.0

5lj5

11.3

0.36

[90]

Prp16 in C Prp22 in C* Prp22 in C*

S.c. H.s. S.c.

EMD-4055 EMD-3541

3.8 5.9 4.17

5lj5 5mqf 5mq0

n.a. n.a. 8.7

n.a. n.a. 0.35

[90] [91] [92]

Brr2 in tri-snRNP

S.c.

EMD-8012

3.7

5gan

7.5

0.31

[93]

Brr2 in tri-snRNP

S.c.

EMD-8013

4.2

5gao

4.2

0.78

[93]

Brr2 in tri-snRNP

S.c.

EMD-8014

3.6

5gap

4.0

0.53

[93]

Brr2 in tri-snRNP

S.c.

EMD-6561

3.8

3jcm

10

0.03

[94]

Brr2 in Bact

S.c.

EMD-9524

3.5

5gm6

3.5

0.74

[89]

Brr2 in Bact

S.c.

EMD-4099

5.8

5lqw

Chain O only CA atoms Chain Y all atoms Chain Q only Poly-Ala Not included Not released Chain V Poly-Ala of 645 residues Chain B all atoms Chain B all atoms Chain B all atoms of 71 residues Chain N all atoms Chain B all atoms Chain O only CA atoms

6.5

0.71

[88]

Brr2 in C

S.c.

EMD-4057

10.0

5lj5

13.5

0.13

[90]

R. Ficner et al. / Methods 125 (2017) 63–69

2.5. Helicase activity assays The most widely used method for studying the helicase activity of spliceosomal DExD/H-box proteins has been the electrophoretic mobility shift assay (EMSA), which allows the separation and quantification of radiolabeled double-stranded and singlestranded RNA after incubation with a helicase. Details of this method and its application to other RNA helicases are described elsewhere [64]. Since gel-based methods are limited to measure the kinetics of fast dsRNA unwinding, fluorescence-based stopped-flow techniques were developed [65,66]. However, this technique has so far not been applied to spliceosomal helicases. Only for Prp43, a fluorescence based assay was used to measure helicase activity [67,68]. Briefly, a dsRNA with 30 ssRNA overhang was used, in which the shorter RNA strand carried a Cy5 fluorophore at the 50 end, and the blackberry quencher BBQ was attached to its 30 end. Upon dsRNA unwinding the released short strand forms an internal hairpin, positioning BBQ and Cy5 in close spatial proximity and leading to a decrease of Cy5 fluorescence due to quenching by BBQ. 2.6. RNA binding The RNA binding properties of some spliceosomal helicases were most frequently analyzed by electrophoretic mobility shift assays (EMSA), a method reviewed in [69]. Alternatively, fluorescence anisotropy was used to determine the binding constants of RNA and spliceosomal helicases. The basics of this method have been described in several reviews, e.g. [66,70,71]. 3. Conclusion High-resolution structures for many spliceosomal helicases are known, however, for Prp2, Prp16, and Prp22 they are still missing. Recent single particle cryo-EM structures of different spliceosomal complexes provided exciting insight into the architecture of the spliceosome and allowed to locate Brr2, Prp2, Prp16 and Prp22 in the spliceosome shedding more light on their respective role in the splicing process. However, due to the low local resolution of the EM maps no refined atomic models of these spliceosomal helicases could be obtained and therefore details regarding conformational changes or protein-RNA interaction could not be derived. There are still open questions regarding fundamental aspects on the function of spliceosomal helicases. The mechanism by which the G-patch protein Ntr1 activates the DEAH-box helicase Prp43 is still an enigma, while for example the closely related DEAHbox protein Prp22 is active on its own. Even more puzzling is the finding that Prp2 in complex with its G-patch protein Spp2 exhibits RNA-stimulated ATPase activity, but lacks any helicase activity. Based on the crystal structure, the DEAH-box protein Prp43 as well as other members of the DEAH-box subfamily were proposed to be processive helicases, however, this has not yet been confirmed experimentally. Acknowledgement We thank the DFG – Germany for funding (SFB860, TP A02 to R.F.). References [1] C.L. Will, R. Lührmann, Spliceosome structure and function, Cold Spring Harbor Perspect. Biol. 3 (7) (2011) 181–203. [2] O. Cordin, D. Hahn, J.D. Beggs, Structure, function and regulation of spliceosomal RNA helicases, Curr. Opin. Cell Biol. 24 (3) (2012) 431–438. [3] O. Cordin, J.D. Beggs, RNA helicases in splicing, RNA Biol. 10 (1) (2013) 83–95.

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