Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

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Article

Reference

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

WEICHENRIEDER, O, et al.

Abstract

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition [...]

WEICHENRIEDER, O, et al . Hierarchical assembly of the Alu domain of the mammalian signal recognition particle. RNA , 2001, vol. 7, no. 5, p. 731-40

PMID : 11350037

Available at:

http://archive-ouverte.unige.ch/unige:17514

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Hierarchical assembly of the Alu domain

of the mammalian signal recognition particle

OLIVER WEICHENRIEDER,^1,4CATHERINE STEHLIN,¹ULRIKE KAPP,¹DARCY E. BIRSE,¹ PETER A. TIMMINS,² KATHARINA STRUB,³ and STEPHEN CUSACK¹

1European Molecular Biology Laboratory,Grenoble Outstation,F-38042 Grenoble Cedex 9,France

2Institute Laue-Langevin,F-38042 Grenoble Cedex 9,France

3Département de Biologie Cellulaire,Université de Genève,Sciences III,CH-1211,Geneva 4,Switzerland

ABSTRACT

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal se- quence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRPAlu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to theAlu RNA 59domain. This creates the binding site for the Alu RNA 39domain.Alu RNA then undergoes a large conformational change with the flexibly linked 39domain folding back by 1808onto the 59 domain complex to form the final compactAlu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to trans- lational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived fromAlu elements in genomic DNA.

Keywords: retroposition; RNA–protein recognition; RNP assembly; signal recognition particle; translational control

INTRODUCTION

The signal recognition particle (SRP;Walter & Blobel, 1980),like the ribosome,is a cytoplasmic ribonucleoprotein particle (RNP) of ancient evolutionary origin (Poritz et al+,1990;Bhuiyan et al+,2000)+SRP has an intrinsic affinity for ribosomes (Walter et al+,1981) and its catalytic promotion of the cotranslational mode of protein translocation across membranes is well docu- mented (Walter & Johnson, 1994; Lütcke, 1995)+ In mammals,SRP consists of the highly base-paired 300- nt-long SRP RNA and six proteins: SRP54, SRP19, and the heterodimers SRP68/72 and SRP9/14 (Fig+1)+

SRP9/14 associates with the terminal sequences of SRP RNA,forming the enzymatically separableAludo- main of SRP (Gundelfinger et al+, 1983),whereas the other proteins together with the central RNA sequence form the S-domain of SRP+High resolution crystal struc-

tures are now available for a number of SRP components:the NG- and M-domains of SRP54 (Freymann et al+,1997;Montoya et al+,1997;Keenan et al+,1998; Clemons et al+, 1999) and the M-domain in complex with helix 8 of SRP RNA (Batey et al+,2000) as well as the free helices 6 (Wild et al+,1999) and 8 (Jovine et al+, 2000) of SRP RNA,free SRP9/14 (Birse et al+,1997), and,most recently,theAludomain with SRP9/14 clamp- ing together in its concave beta-sheet the 59 and 39 domains of Alu RNA (Weichenrieder et al+, 2000)+ In electron micrographs the particle appears as a flexible, tri-segmented rod of 60 Å by 260–280 Å with the two domains distinguishable at opposite ends (Andrews et al+, 1985,1987)+

SRP selects ribosomes displaying the N-terminal signal sequence of nascent secretory and membrane proteins that first emerges at the exit pore on the large ribosomal subunit+TheAludomain of SRP is responsible for retarding the elongation of these proteins once their export signal sequence is bound by the S-domain of SRP and prior to engagement with the translocation machinery in the endoplasmic reticulum+Considering the apparent length of the particle, it has been pro-

Reprint requests to:Stephen Cusack,European Molecular Biology Laboratory,Grenoble Outstation,B+P+156X,F-38042 Grenoble Ce- dex 9,France;e-mail:cusack@embl-grenoble+fr+

4Present address:The Netherlands Cancer Institute,Plesmanlaan 121,1066 CX Amsterdam,The Netherlands+

RNA(2001),7:731–740+Cambridge University Press+Printed in the USA+

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posed that the Alu domain might reach the cleft between the two ribosomal subunits where the elongation factors bind (Andrews et al+, 1987; Siegel & Walter, 1988),but theAluRNP crystal structure does not support the hypothesis that elongation arrest is caused by a mechanism of mimicry-based active competition with

elongation factors (Weichenrieder et al+,2000)+Knowl- edge of the preferred orientation and the degree of flexibility of theAludomain (and the crucial C-terminal tail of SRP14;Thomas et al+,1997;Mason et al+,2000) with respect to the signal sequence binding S-domain will therefore be of primary importance for an explana- tion of the mechanism of SRP-mediated elongation arrest+ These features, which result from the proper assembly of the particle, are decisive in determining the physiologically relevant size and shape of SRP and they govern possible multivalent interactions of SRP with different regions of the ribosome+

The assembly of the S-domain of SRP has been found to be cooperative in vitro (Walter & Blobel,1983) and to partially take place in the nucleolus and in the cytoplasm in vivo (Politz et al+, 2000)+This underlines the notion that the assembly of multicomponent particles like SRP or the ribosome in vivo has to be imag- ined in a stepwise, hierarchical, and controlled order with precise temporal and spatial separation of events+ Concerning the assembly and function of theAlu domain of SRP it has been shown previously that SRP9/14 heterodimerization is a prerequisite forAluRNA binding (Strub & Walter,1990)+We have also determined experimentally the minimal Alu RNA folding domain, SA86 (Weichenrieder et al+,1997),which consists of a distinct 59domain (nt 1– 47 of human SRP RNA) co- valently linked to a 39 domain (nt 48– 64 of SRP RNA base paired to nt 282–300;Fig+1)+AluRNA constructs designed according to SA86 have been shown to be sufficient for accurate SRP RNA 39 end processing (Chen et al+,1998) and for efficient export of SRP RNA to the cytoplasm (Jacobson & Pederson,1998)+A construct corresponding to the SA86 59 domain strongly activates transcription by RNA polymerase III (Emde et al+, 1997)+

Structures with very similar RNA and protein components to the SRPAludomain are found in other,non- SRP contexts (Fig+1) and understanding the assembly and conformational state of theseAlu RNPs will also be critical to understanding their function+ Most interesting are the neuron-specific BC200 RNP,which mi- grate into dendrites to possibly regulate localized protein translation (Kremerskothen et al+, 1998) and the Alu retroposition intermediates repeated in tandem,which are responsible for the creation of 10–12% of the human genome in the form ofAluelements (Mighell et al+, 1997)+Aluretroposition is an ongoing process that must have had a significant impact on the evolution of the human genome (Kazazian,1998)+

Here we delineate in vitro the detailed assembly pathway for the SRPAludomain by studying the behavior in solution of variants of its RNA and protein components+We discuss the physiological implications of the results in the light of the crystal structures,which have been determined of two Alu RNP variants (Weichen- rieder et al+, 2000)+

FIGURE 1. Schematic representations of mammalian SRP and re- latedAluRNPs+TheAludomain is colored red (SRP9),green (SRP14), blue (AluRNA 59domain),and cyan (AluRNA 39domain)+Other parts with a known crystal structure are orange+A: Mammalian SRP consisting of SRP RNA bound to six proteins+B: HypotheticalAlu retroposition intermediate consisting of twoAlusequences arranged in tandem each bound to SRP9/14 and of a long poly-A tail+Small cytoplasmicAluRNPs are thought to be processed derivatives thereof corresponding to the left arm complex+C: The neuron-specific BC200 RNP consisting of SRP9/14 bound to a monomericAluRNA with a unique 39terminal sequence+D: SA86,the minimalAluRNA required to bind SRP9/14 with full specificity+The S domain is artificially replaced by a GUAA tetraloop (gray)+ The natural positions of the RNA 59and 39ends allow for considerable flexibility between the RNA 59 and 39domains (green arrow)+ RNase V1 cleavage sites protected upon addition of SRP9/14 are colored green,continuously accessible sites red+H:helix;L:loop+

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RESULTS

Preferential binding of SRP9/14 to the 59domain of AluRNA as the primary recognition event

In gel filtration experiments,under conditions that would allow reconstitution of completeAluRNPs with SA86, SRP9/14 binds only to 59domain RNA (SA47,blue in Fig+1D) even if 39domain RNA (SA39,cyan in Fig+1D) is present in trans in equimolar amounts (Fig+2A)+Spec- ificity of the interaction of the 59domain with SRP9/14 is indicated by the comparison of RNase V1 digestion

patterns of variousAluRNAs in the absence and presence of SRP9/14+The protection of nt G20 to C22 from cleavage (Fig+ 2B,C) is typical and agrees well with hydroxyl-radical footprinting data on complete SRP RNA (Strub et al+,1991)+The continued exposure and cleavage at A43 is characteristic as well,suggesting that the first and third stem stack in the complex+ The slight enhancement of cleavage at this site in the presence of protein,which is even more pronounced in the context of longerAluRNAs,suggests that SRP9/14 stabilizes the stacked conformation+

These results,obtained in solution,are entirely consistent with the crystal structure of a specificAluRNP consisting of SRP9/14 bound to 59 domain Alu RNA (SA50,Fig+2B;Weichenrieder et al+,2000)+In solution, the free SA50 is likely to be largely prefolded but with enhanced flexibility between the helical stacks to the extent that the tertiary interactions between the two loops (including three Watson–Crick base pairs) may be partially disrupted (Figs+2C,4B)+Specific binding of SRP9/14 to the conserved U-turn of the 59 domain rigidifies the structure and promotes formation of the tertiary interactions+Such flexibility between helical RNA stacks is of general importance for the process of RNA folding and might be functionally relevant in many cases (Batey & Doudna,1998)+In summary,the preferential binding of SRP9/14 to the Alu RNA 59 domain is a novel observation and the structural rearrangements that occur in the RNA upon protein binding illustrate the role of proteins to assist large RNAs in adopting their physiologically relevant conformations+

Detection of a secondary recognition event in the context of a flexibly linkedAluRNA 39domain

To investigate further the role of the RNA 39domain in protein binding, we made use of two circularly per- mutatedAluRNAs,SA88 and SA91,in which the original 59and 39ends of SA86 are connected with linkers of one or four uridines, respectively+ In SA88, the 39 domain is designed to stack on the terminal stem of the 59domain,whereas in SA91,due to the bulge of three uridines,it has considerably more flexibility,mimicking wild-type RNA (Fig+3A,B)+We first compared the relative affinities of SRP9/14 for SA86,SA91,and SA88 in a pilot competition experiment based on nitrocellulose filter binding using radiolabeled SA86 and com- peting with increasing concentrations of unlabeled SA86, SA91,and SA88+The titration experiment (Fig+3C,D) indicates SA88 to have about 50–100-fold lower affinity for SRP9/14 than SA86+

Based on these results,we then did gel retardation experiments with radiolabeled SA86, SA91, or SA88 and a 20-fold excess of each SA86,SA91,SA88,and SA50 as unlabeled competitor RNA+Under these conditions we can confirm that SA86 and SA91 have vir-

FIGURE 2. Preferential and specific binding of SRP9/14 to theAlu RNA 59domain (in A,particle concentrations are below 5mM;in C, 500 nM)+ A: Analytical gel filtration chromatography using sub- fragments SA86+SA47 (blue in Fig+1D) corresponds to the 59do- main of SA86 and SA39 (cyan in Fig+1D) corresponds to the 39 domain of SA86+In an equimolar mixture of SA47 (blue profile) and SA39 (cyan profile) with a limiting amount of SRP9/14, complex formation takes place (black profile,peak 1)+Analysis of the peak fractions (insert) on a denaturing RNA gel reveals peak 1 to contain only SA47 RNA+In the peak of free RNA (black profile,peak 2) SA39 is in excess+V:Elution volume;OD:Optical density+B: SA50,theAlu RNA 59domain stabilized by two additional base pairs (gray)+C: En- zymatic probing with double-strand-specific RNase V1+Specific interaction of theAluRNA 59domain (SA50) with SRP9/14 is indicated by the protection of diagnostic cleavage sites at G20-C22 (green) and the enhancement of cleavage at A43 (red)+L:nucleotide ladder;

G:guanosine ladder+

Alu RNP assembly 733

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tually identical protein affinity (Fig+4A,lanes 4 and 10), whereas SA88 apparently has a significantly lower one (Fig+ 4A, lanes 5 and 12)+ Furthermore, the efficient competition of SA50 with SA88 but not with SA86 or SA91 suggests that in the SA88 RNP the protein heterodimer is exclusively bound to the AluRNA 59 domain (Fig+ 4A, lanes 6, 13, and 20)+ Enzymatic RNA probing experiments in the presence and absence of SRP9/14 support this interpretation+RNA helix H3+3 is protected from RNase V1 cleavage only if the link to the Alu RNA 59 domain is flexible (Fig+ 4B,C)+ This correlation suggests that the higher specificity of SA86 and SA91 for SRP9/14 results from a second recognition event between the Alu RNA 39 domain and the initially and preferentially formed Alu59 domain RNP+ The use of the rigidifiedAluRNA mutant,SA88,allows us to experimentally separate the two binding events+ As a consequence, the SA88 Alu RNP should have accessible binding surfaces that might lead to dimerization or multimerization of complexes at elevated particle concentrations+

A compactly folded RNP resulting from a large-scale protein-induced AluRNA bending

To study the biophysical properties and the stoichiom- etry of freeAluRNAs and AluRNPs in concentrated solution we analyzed and purified them by preparative gel filtration chromatography for use in small angle neutron scattering experiments (Fig+5)+Neutron small angle scattering allows the independent determination of the molecular weight and the radius of gyration of a particle+For the SA91 and SA88 RNPs (in H₂O buffers) we measured values of relative molecular weight corresponding to 0+99 and 1+96 times the mass of monomeric SA86 RNP+ The corresponding values for the radius of gyration of the SA86,SA91,and SA88 RNPs are 27+7 (60+5), 28+2 (60+5), and 37+0 (61) Å+ The results strongly suggest that under these conditions the SA88 RNP forms mainly dimers,whereas the SA86 and SA91 RNPs are monomers+ In contrast, the free RNAs are monomeric in all three cases because the measured relative molecular weights for SA91 and SA88 RNA are 1+12 and 1+01 times the value of monomeric SA86 RNA+Similar results are found by preparative gel filtration of complexes (particle concentration above 100mM):the SA88 RNP has an anomalously high hydrodynamic radius compared to the SA91 and the SA86 RNPs,whereas all three RNAs behave similarly+In the gel retardation assay,where particle concentrations are below 5 mM, dimerization of the SA88 RNPs is not detected,pointing to a rather weak interaction+

The observation that dimer formation of the SA88 RNP in solution is clearly a property of the entire ribonucleoprotein particle,but not of its individual compo-

FIGURE 3. Contribution of a flexibly linkedAluRNA 39domain to protein affinity+(particle concentrations are 500 nM) A: SA91,a circular permutation of SA86+The original 59and 39ends are connected by a tetra-uridine linker (magenta) that keeps considerable flexibility between the RNA 59and 39domains (green arrow)+The GUAA tetraloop is replaced by a terminal stem (grey)+B: SA88,a circular permutation of SA86 identical to SA91 except for a mono-uridine instead of a tetra-uridine linker+TheAluRNA 59and 39domains are con- strained to be in a stacked,extended conformation indicated by the red bar+C: Equilibrium competition experiments with labeled SA86 analyzed by nitrocellulose filter binding+The autoradiograph of the multislot filter membrane shows SA88 to compete with SA86 for SRP9/14 only at elevated ratios,r,of competitor to labeled RNA+

SA160: extendedAlu RNA construct (Strub et al+,1991); SS125:

nonspecific SRP S domain RNA+D: Quantitation of C withn,the fraction saturation of SRP9/14 as a function ofr(Weichenrieder et al+,1997)+The solid curves correspond to competitors with 1-,10-, 100-,or 1000-fold higher dissociation constants than SA86+ Sub- sequent competition experiments were done atr520 (dotted line)+

Symbols are as for C+

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nents is explained by the crystal structure of the SA88 RNP (Weichenrieder et al+, 2000)+ In the crystals,dimers of the SA88 RNP are observed that arise from the interaction of theAluRNA 39domain of one SA88 RNP with theAluRNP 59domain of a second,crystal- lographically twofold related, SA88 RNP+ This result, together with the protection of theAluRNA 39domain from RNase V1 cleavage in the monomeric SA86 and SA91 RNP complexes,leads to a model of the compact AluRNP monomer in which the 59 domain RNP interacts with its own 39 domain RNA (Weichenrieder et al+, 2000; Fig+ 7)+ Because the neutron scattering and gel filtration experiments suggest the free SA86, SA91,and SA88 RNAs all adopt similar conformations (probably resembling the extended conformation ob-

served in the SA88 RNP monomer),the model of the compactAluRNP monomer implies a large conformational change ofAluRNA upon complete SRP9/14 binding,turning the 39domain by 1808+

Corroboration of the compact AluRNP model by mutagenesis of conserved amino acids

The possibility to experimentally distinguish in the gel retardation assay between the strong interaction of SRP9/14 with the Alu RNA 59 domain and the weak interaction of theAluRNP 59domain with theAluRNA 39 domain allowed us to further corroborate the sug-

FIGURE 4. Experimental distinction between the primary (59domain) and secondary (39domain) binding sites of SRP9/14 onAluRNA+A: Relative affinities of variousAluRNA constructs for SRP9/14 as revealed by an electrophoretic mobility shift assay+Direct competition experiments show SA86 and SA91 to have similar protein affinities,whereas SA88 can be competed out with isolated 59domain RNA (SA50)+SS125:nonspecific SRP S domain RNA+B: Specific interaction of SRP9/14 with SA91 as revealed by enzymatic probing with double-strand-specific RNase V1 (V1)+In the presence of SRP9/14,diagnostic cleavage sites at G20-C22 and at U60 and C61 get protected (green in Fig+3A),whereas cleavage at A43 gets enhanced (red in Fig+3A)+L:nucleotide ladder;G:guanosine ladder+C: Protection of theAluRNA 39domain from RNase V1 cleavage in SA91AluRNPs versus exposure in SA88AluRNPs+

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gested compact Alu RNP model by mutation of conserved amino acids in the RNA protein interface+Our rationale was that mutations of amino acids involved in AluRNA 59domain binding should weaken complexes with labeled SA91 but not change their resistance towards competition with cold SA50 competitor+In contrast,mutations of amino acids involved in the bending of theAluRNA 39domain or in the stabilization of the bent RNA conformation should lead to more open, slower migrating complexes that are susceptible to cold SA50 competitor,very much like complexes of labeled SA88 with wild-type SRP9/14 (Fig+ 6)+ We therefore tried to identify such a case in support of the compact AluRNP model+

All of the single or double point mutations of SRP9/14 that we tested still form specific complexes with SA91 AluRNA (Fig+ 6A)+Among these,the strongest RNA- binding defects are detected for the SRP9 K30A/R32A double mutant and the SRP14 R59A substitution,where side chains critical for the specific recognition of the 59 domain have been replaced (Weichenrieder et al+,2000)+ The other single point mutants of SRP9 (C39S,C48S, E15A) or SRP14 (Y27A) do not behave significantly differently from wild-type protein+This is in agreement with the structural model (Weichenrieder et al+,2000), where the respective side chains are not part of the RNA–protein interface at all (SRP9 E15A),part of the interface but not within hydrogen bonding distance (SRP9 C39S),or potentially affecting only a single weak contact to theAluRNA 59domain (SRP14 Y27A) or 39 domain (SRP9 C48S)+

The SRP9 K41A/C39A double mutant displays the most interesting phenotype (Figs+ 6A and 6B)+Its se- lective deficiency inAluRNA 39domain binding (Fig+6) is most probably due only to the substitution of SRP9 K41, because SRP9 C39 is located rather far away (.5+5 Å) from the RNA in the structure and its substitution with serine has no effect (Fig+6A)+The complex of SA91 with the mutant SRP9/14 is susceptible to SA50 competition and migrates slightly slower in the gel than the complex with wild-type SRP9/14+This indicates a more open conformation of this mutant RNP+ The mutation does not seem to affect 59domain binding as reflected by the migration profiles of the respective wild-type and mutant complexes with the rigidified SA88AluRNA and their behavior towards competitor RNAs (Fig+ 6B)+ SRP9 K41 is one of three potential candidates (SRP9 K41, SRP9 D45, SRP9 C48) sug- gested by the 4 Å crystal structure to affectAluRNA 39 domain binding+The side chain of SRP9 K41 is posi- tioned between the 59and 39domain RNA backbones (Fig+6C),consistent with an important function in stabilizing the bent RNA conformation+Thee-amino nitro- gen of K41 is close to the 59domain (O39of U23) and 39domain (O29of G58) nucleotides although the cur- rent resolution does not permit a precise definition of its interactions+

FIGURE 5. Dimerization of soluble SA88AluRNPs at high particle concentrations (SA86 (black), SA91 (green), SA88 (red); particle concentrations above 100mM)+A: Preparative gel filtration chroma- tography+AluRNAs (thin lines) are of similar size and shape,whereas the SA88AluRNP has a considerably larger hydrodynamic radius than its counterparts (thick lines)+ V: Elution volume; OD: optical density;*:excess protein+B: Neutron small angle scattering of un- complexed Alu RNAs+A Guinier plot of representative scattering curves normalized for concentration (;2+5 mg/mL) and for the molecular weight of SA86AluRNA reveals almost identical masses and radii of gyration+C: Neutron small angle scattering ofAluRNPs+The Guinier plot, normalized for concentration (;9+0 mg/mL) and the molecular weight of the SA86AluRNP clearly shows the SA88Alu RNP to form dimers with a significantly larger radius of gyration+

q: scattering vector;I(q):scattering intensity+

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DISCUSSION

Hierarchical assembly ofAluRNPs in vitro

Taking into account the presented biochemical data and previous structural data on the SRPAlu domain (Weichenrieder et al+, 2000),we deduce a sequential assembly pathway for the SRP Alu domain in vitro (Fig+7)+In the first step,SRP9 and SRP14 must heterodimerize,because neither of them alone bindsAlu

RNA (Strub & Walter, 1990)+ In the second step the SRP9/14 heterodimer associates with free Alu RNA, which is monomeric and likely to be in an extended conformation,because the measured radii of gyration of SA86,SA91,and SA88 are all similar+SRP9/14 initially interacts with the Alu RNA 59 domain, inducing and/or stabilizing the stacking of RNA helices H1+2 and H1+1 and possibly strengthening the tertiary interactions between loops L2 and L1+2+At this intermediate stage in assembly the particle might resemble the rigidified

FIGURE 6. Analysis of various SRP9/14 point mutations with respect toAluRNA binding+A: Screening of seven mutants at equal total protein concentration in an electrophoretic mobility shift assay+M1:SRP9 E15A;M2:SRP9 K30A/R32A;M3:

SRP9 C39S;M4:SRP9 C39A/K41A;M5:SRP9 C48S;M6:SRP14 Y27A;M7:SRP14 R59A+All mutants form specific complexes with bendable SA91AluRNA,although to a different extent+Only M4 is differentially affected by SA50 competition+B: Detailed analysis of the defect of M4 inAluRNA 39domain binding+Note the slightly slower migration of the

SRP9^M4/14-SA91AluRNP+C: Stereo diagram of the proposedAluRNP structure (Weichenrieder et al+,2000) viewed down

the SRP9/14 (red/green)b-sheet and illustrating the distinguished position of SRP9 K41 that allows it to contact both the RNA 59domain (blue) and the RNA 39domain (cyan)+Occluded and accessible RNAse V1 cleavage sites are shown in green and red+

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monomeric SA88 RNP+In the third step of the assembly pathway,the flexibly linkedAluRNA 39domain flips like a jackknife to contact a surface on the 59domain complex,which is composed of both SRP9 and 59do- main RNA+ We identified SRP9 K41 as an essential residue for the formation of the closed conformation+ Concomitant to this final and substantial conformational change,the RNA helix H3+3 gets protected from nuclease attack and the natural RNA 39 end gets ex- posed+An important consequence of this model is that the stem leading to the SRP S-domain emerges on the SRP14 C-terminal side of SRP9/14,and points in the diametrically opposite direction to that it would if there was no RNA bending+ This discussion highlights the

truly sequential order of the assembly process,as the formation of the 59domain complex is a prerequisite for the binding of the RNA 39domain as is SRP9/14 heterodimerization forAluRNA 59domain binding+

The dissociation constant of SRP9/14 and SRP RNA is smaller than 0+1 nM (Janiak et al+,1992),corresponding to a total free binding energy of more than 13+7 kcal/mol, and our preliminary estimates indicate that the closed conformation is only about 2+3–2+7 kcal/mol more stable than the open conformation+ This raises the questions of how readily reversible is the conformational switch between the open ((46 Å3 25 Å)3 95 Å in length) and closed ((46 Å350 Å)3 59 Å in length)AluRNA and whether the switch could be trig- gered in the physiological context of SRP interacting with other cellular components such as the ribosome+ There is experimental support for the reversibility of 39 domain binding in the flexibly linked SA86 RNP, because purified compact monomers are found to crys- tallize as SA88 RNP-like domain-swap dimers of the open form (O+Weichenrieder and S+Cusack, unpubl+ data), presumably favored by the high concentration+ Also in the context of purified,complete SRP,there are experimental conditions under which the SRP9 cys- teines are preferentially accessible to alkylation, indi- cating an opening of theAludomain (Walter & Blobel, 1980)+

Physiological aspects ofAluRNP assembly and 59-39domain flexibility

The assembly pathway of theAluRNP in vivo is likely to be the same as in vitro but must be seen in the context of the complete SRP and in the context of the compartmentalization of the eukaryotic cell,which leads to a spatial separation of events+

Combining our results on the assembly of the Alu domain with previously published data and hypoth- eses,we suggest the following scenario for the assembly of SRP in vivo+SRP9 and SRP14 first heterodimerize in the cytoplasm and subsequently enter the nucleo- plasm+There SRP9/14 binds cotranscriptionally to the AluRNA 59domain enhancing transcriptionin cis(Emde et al+, 1997)+After termination of transcription,theAlu RNA 39 domain bends into the closed conformation and exposes the 39end for nuclear processing (Chen et al+, 1998)+ Then SRP19 and SRP68/72 join the S-domain RNA in the nucleolus before the subparticle gets exported to the cytoplasm where SRP54 adds on in a final step (Politz et al+,2000)+There is experimental evidence that Alu RNA contains all necessary elements for efficient nuclear export (Jacobson & Peder- son,1998)+Although the presence of SRP9/14 in the nucleus and its cotranscriptional association with Alu RNA have not explicitly been demonstrated, we con- sider the scenario to be quite plausible+ Recent data from the distantly related yeast (y)SRP (Ciufo & Brown,

FIGURE 7. Hierarchical assembly pathway of the SRPAludomain+

Heterodimerization of SRP9 (red) and SRP14 (green) is a prerequisite (Strub & Walter,1990) forAluRNA 59domain (blue) binding+This interaction induces the RNA 39domain (cyan) to flip around and bind across the interface of the 59domain complex+The model is consistent with the RNase V1 probing data (light green and light red phosphate–ribose backbone) and explains the role of SRP9 K41 (violet) in 39domain binding (contacted oxygens in yellow)+At very high particle concentrations,an intermolecular 59domain–39domain interaction could be favored,leading to particle dimerization+

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2000) show that ySRP14 can be detected in the nucleolus where it takes part in the assembly of a core particle together with ySRP21,ySRP68,ySRP72,and ySRP RNA (scR1)+ The model presented above is equally likely to be valid for the assembly, transport, and function of non-SRPAluRNPs+

The potential flexibility of the Aludomain might be relevant for the elongation arrest function of SRP+ It would allow SRP to exist in clearly different states dur- ing its functional cycle and it would pose the crucial C-terminal tail of SRP14 (Thomas et al+,1997;Mason et al+, 2000) into alternating structural environments+ In the context of the Alu retroposition intermediates repeated in tandem,the flexibility between theAluRNP 59and 39domain combined with the high local particle concentration due to the covalent linkage could favor the formation of a compact particle that resembles the SA88 RNP dimer+Such a particle could be of functional importance at some stage in the retroposition cycle+

MATERIAL AND METHODS

Preparation of RNA and protein samples

RNA molecules with 39terminal hammerhead ribozymes were synthesized by in vitro transcription with T7 RNA polymerase from HindIII linearized plasmid templates pSA86H(2), pSA91H, pSA88H(2), and pSA50H (Weichenrieder et al+, 2000)+ Products were purified as described (Weichenrieder et al+,1997)+Plasmids pSA47H and pSA39H,coding for SA47 and SA39, were obtained by introducing synthetic oligonu- cleotides into plasmid pSA86H (Weichenrieder et al+,1997) digested withAvaI/HindIII or EcoRI/XbaI, respectively+Hu- man SRP9^1–86/14^1–108 (referred to as SRP9/14) was obtained as described (Weichenrieder et al+, 2000)+ Mutants thereof were made with the QuickChange (Stratagene) site- directed mutagenesis protocol,using for SRP9 a derivative of pQE-70 (Quiagen) as the parent vector andEscherichia coli M15 (pREP4) cells for expression+Mutant SRP9 and SRP14 proteins,after the heparin ion exchange chromatography purification step, were combined with unmutated partners to form mutant heterodimers,except for SRP9 K30A/R32A and SRP9 C39A/K41A mutants,which were copurified as heterodimers with wild-type SRP14^1–108+

RNA competition experiments and enzymatic probing of RNA structure

Competition experiments were done and quantitated as described previously (Weichenrieder et al+, 1997)+ Samples (20 mL, 20,000 cpm) for comparing the relative affinities of variousAlu RNA constructs for SRP9/14 contained 500 nM of each SRP9/14 and 59³²P-labeled substrate RNA and 10mM of unlabeled competitor RNA+RNAs were preannealed and complex formation was analyzed by RNA mobility shift in nondenaturing 8% polyacrylamide gels+For the screening of protein mutants,200 nM labeled SA91 RNA was mixed with 2mM total protein and challenged with 4mM SA50 compet-

itor RNA+ The SRP9 C39A/K41A mutant was further analyzed in the context of 200 nM labeled substrate RNA, an equal amount of active protein,and 4mM of the respective competitor RNA+Samples (25mL,100,000 cpm) for RNase V1 probing (Weichenrieder et al+,1997; 0+2–0+7 enzymatic units for 2–5 min at 378C) contained 500 nM 59³²P-labeled Alu RNA,10mM SRP9/14,and 0+2 mg/mL ofEscherichia coli tRNA+

Gel filtration chromatography and neutron small angle scattering

For analytical purposes (Fig+2),1 mL of pre-annealed 6mM RNA(s) and 4mM SRP9/14 was analyzed on a HiLoad 16/60 Superdex 200 column (Pharmacia)+ For preparative purposes (Fig+5),particles were purified from a starting mixture (1 mL) of 150mMAlu RNA and 300mM SRP9/14 and concentrated to 20 mg/mL+ Neutron small angle scattering experiments were done on instrument D22 of the Institut Laue Langevin,Grenoble+Samples (2+3–22+2 mg/mL in buffers of various H2O/D2O ratios) were measured at a wavelength of 10 Å,a detector distance of 3 m and in cells with 0+1 or 0+2 cm path length (1 cm² illuminated area)+For each sample, the transmission,T,of the direct beam and the intensityI (q) of the scattered radiation as a function of the scattering vector, q,were determined+Data,normalized using scattering from pure H2O, was plotted according to the Guinier equation, lnI (q)5lnI (0)2(RG2

/3)q²,whereRGis the radius of gyration and I (0) the scattering intensity at zero angle that can be used to calculate the molecular weight as described (Jacrot & Zaccai,1981)+

Structure coordinates and figures

PDB entry codes are le80 for the SA50 RNP and le8s for the SA88 RNP (Weichenrieder et al+,2000)+ Figures were pro- duced with BobScript (Esnouf, 1997)/Raster 3D (Merritt &

Bacon,1997)+

ACKNOWLEDGMENTS

P+T+did the neutron small-angle scattering experiments+D+B+ provided expression clones for SRP9 point mutants, C+S+ made expression clones for SRP14 point mutants and pre- pared mutant proteins together with U+K+ O+W+ received a predoctoral fellowship from the Boehringer-Ingelheim-Fonds+ K+S+is supported by a START subsidy and a research grant from the Swiss National Science Foundation and the Canton de Genève+ K+S+ and S+C+ are both principal investigators belonging to SRPNET,a research network of the European Union Training and Mobility in Research program+

Received January 30, 2001; accepted without revision February 19, 2001

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