Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation

Article

Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation Graphical Abstract

Authors Valentina Ahl, Heiko Keller, Steffen Schmidt, Oliver Weichenrieder

Correspondence [email protected]

In Brief Ahl et al. determined the crystal structure of a conformationally closed Alu ribonucleoprotein particle that matters for the signal recognition particle and the retrotransposition of the human Alu element. A single Alu folding unit is sufficient for retrotransposition activity. It likely targets stalling ribosomes to recruit nascent L1 reverse transcriptase.

Highlights d

Identification of a minimal Alu RNA sufficient for retrotransposition

d

High-resolution crystal structure of a human Alu RNP in the closed conformation

d

An ancient Alu RNA structural core in SRP RNAs interfaces the ribosome

d

Targeting stalled ribosomes could overcome L1 cispreference

Ahl et al., 2015, Molecular Cell 60, 1–13 December 3, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.10.003

Accession Numbers 5AOX

Please cite this article in press as: Ahl et al., Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.003

Molecular Cell

Article Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation Valentina Ahl,1 Heiko Keller,1,2 Steffen Schmidt,1 and Oliver Weichenrieder1,* 1Department

of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tu¨bingen, Germany address: Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.10.003 2Present

SUMMARY

The Alu element is the most successful human genomic parasite affecting development and causing disease. It originated as a retrotransposon during early primate evolution of the gene encoding the signal recognition particle (SRP) RNA. We defined a minimal Alu RNA sufficient for effective retrotransposition and determined a high-resolution structure of its complex with the SRP9/14 proteins. The RNA adopts a compact, closed conformation that matches the envelope of the SRP Alu domain in the ribosomal translation elongation factor-binding site. Conserved structural elements in SRP RNAs support an ancient function of the closed conformation that predates SRP9/14. Structurebased mutagenesis shows that retrotransposition requires the closed conformation of the Alu ribonucleoprotein particle and is consistent with the recognition of stalled ribosomes. We propose that ribosome stalling is a common cause for the cis-preference of the mammalian L1 retrotransposon and for the efficiency of the Alu RNA in hijacking nascent L1 reverse transcriptase.

INTRODUCTION The Alu retrotransposon (Figures 1 and S1) (Deininger et al., 1981) is the prevalent mobile genetic element in humans (Burns and Boeke, 2012; de Koning et al., 2011; Hancks and Kazazian, 2012; Lander et al., 2001). During primate evolution, it colonized the genome in several waves that are reflected by distinct Alu families. Of these families, AluJ is the oldest and now extinct, and AluY is the youngest and is still active (Figure S1A) (Batzer and Deininger, 2002; Bennett et al., 2008; de Koning et al., 2011; Hormozdiari et al., 2013; Lander et al., 2001). In total, the human genome harbors 1.1 million Alu elements, which account for
11% of its sequence and include several thousand potential source genes (Bennett et al., 2008; de Koning et al., 2011; Hancks and Kazazian, 2012; Lander et al., 2001). Alu retrotransposition occurs in the germline and during early embryogenesis (Macia et al., 2011), contributing to genetic het-

erogeneity in the human population (Stewart et al., 2011; Witherspoon et al., 2013). Retrotransposition also occurs in somatic tissues, especially during neuronal development (Baillie et al., 2011; Upton et al., 2015) and in numerous cancer cells (see Shukla et al., 2013 and references therein), although there are efficient silencing mechanisms at multiple steps of the retrotransposition cycle to limit the impact of this mutagenic process (Ade et al., 2013; Burns and Boeke, 2012; Heras et al., 2014; Levin and Moran, 2011). Alu elements spread via Alu RNA retrotransposition intermediates that are transcribed by RNA polymerase III (Pol III) and consequently contain 50 -proximal A-box and B-box sequences as a result of the internal promoter (Perez-Stable and Shen, 1986). Alu retrotransposition intermediates also contain a genome-encoded 30 -oligo(A) sequence that is thought to recruit the reverse transcriptase (RT) encoded by the mammalian L1 retrotransposon (Boeke, 1997; Dewannieux et al., 2003; Jurka, 1997; Okada et al., 1997). The L1-RT permits Alu RNAs to complete the retrotransposition cycle via ‘‘target-primed reverse transcription’’ into a DNA copy at a new genomic location (Figure S1B) (Cost et al., 2002; Dewannieux et al., 2003; Luan et al., 1993). Alu elements are found exclusively in primates, and they are unique among small, Pol III-dependent retrotransposons because they are derived from 7SL RNA and not from a tRNA (Okada et al., 1997). 7SL RNA forms the scaffold of the signal recognition particle (SRP), a ubiquitous cytoplasmic ribonucleoprotein particle (RNP) that interacts with the ribosome to control the translation and export of membrane and secretory proteins (Nyathi et al., 2013; Walter and Blobel, 1982; Wild et al., 2002). Alu retrotransposition intermediates are derived from SRP RNA by a deletion of the central S domain, followed by a tandem duplication of the remaining Alu domain sequences and the acquisition of the oligo(A) tail (Figures 1 and S1A) (Kriegs et al., 2007). In the SRP, the Alu domain sequences assemble with the SRP9/14 protein heterodimer into a complex Alu RNP structure (Weichenrieder et al., 2000, 2001). Alu retrotransposition intermediates consequently harbor two potential folding units, which are termed the left and right half monomers. On the basis of reconstructed Alu RNA family consensus sequences, each of the monomers has retained the ability to bind SRP9/14 as an apparent requirement for retrotransposition (Bennett et al., 2008). Importantly, the Alu domain of the SRP enters the ribosomal translation elongation factor-binding site, where it interferes Molecular Cell 60, 1–13, December 3, 2015 ª2015 Elsevier Inc. 1

Please cite this article in press as: Ahl et al., Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.003

Alu RNA (AJ)

Alu domain

5’

S domain

Scheme of Alu RNA compared with mammalian SRP. The left and right half Alu RNA monomers each contain a 50 -domain (slate) and a 30 -domain (cyan). The crystallization loop (c-loop) is in gray. Retrotransposition competence of truncated Alu retrotransposition intermediates is indicated relative to reconstructed AluJ RNA (AJ). See also Figures S1 and S2 and Table S1.

SRP9

A-box

right

5’ 3’ SRP14

100%

AAAAAn3’

B-box

B-box

c-loop

5’

5’

5’ AAAAAn3’

111%

A-box

A-box

AJL-H33Δ(B)

AJL-H33Δ

AJL

AAAAAn3’

11%

A-box

left

Figure 1. Identification of a Minimal Alu RNP Retrotransposition Intermediate

SRP

B-box

with ongoing protein translation. This can lead to an ‘‘elongation arrest’’ after the S domain of SRP has recognized the nascent N-terminal signal sequence of the respective membrane or secretory protein that emerges from the tunnel exit of the large ribosomal subunit (Halic et al., 2004; Nyathi et al., 2013). Ribosome binding has also been proposed as an explanation for the ability of the Alu retrotransposition intermediates to overcome the cis-preference (Wei et al., 2001) of the L1-RT for its own encoding L1 RNA. According to this ‘‘ribosome-binding hypothesis,’’ a conserved, SRP-like Alu RNP structure could allow the Alu RNA to bind to ribosomes, where the Alu RNA could hijack the nascent L1-RT in competition with the encoding L1 RNA (Boeke, 1997). To test this model, we therefore investigated whether a minimal single SRP-like Alu RNA folding unit is sufficient to promote retrotransposition, whether it adopts a structure that permits ribosome binding, and whether a ribosome-binding conformation is required for retrotransposition. In particular, it was unclear whether or not the Alu RNP would adopt a so-called closed conformation, for which the helical Alu RNA 30 -domain has to fold back onto the complex Alu RNA 50 -domain like a jack-knife (Weichenrieder et al., 2000, 2001). Back-folding occurs in the context of the mammalian SRP as observed by single-particle reconstruction cryo-electron microscopy (cryo-EM) of the SRP on stalled ribosomes (Halic et al., 2004; Voorhees and Hegde, 2015). We determined the high-resolution structure of a minimized, retrotransposition-competent Alu RNP, which had crystallized in the closed conformation, and therefore revealed the molecular basis of how the Alu RNA 50 -domain interacts with the Alu RNA 30 -domain. The crystal structure also showed how the closed Alu RNP conformation is generally achieved in eukaryotic SRP RNAs and permitted a cross-kingdom comparison with archaeal and bacterial Alu RNA structures (Bousset et al., 2014; Kempf et al., 2014) that suggests a conserved ribosome binding mode. Finally, we used structure-based mutagenesis and functional assays to demonstrate that the closed conformation is indeed required for an efficient retrotransposition and that Alu retrotransposition intermediates have retained the structural determinants to bind stalled ribosomes despite the lack of an SRP-like S domain. Taken together, our analysis strongly supports the ribosomebinding model for Alu retrotransposition, including ribosome 2 Molecular Cell 60, 1–13, December 3, 2015 ª2015 Elsevier Inc.

AAAAAn3’

83%

stalling as an explanation for the high efficiency of the retrotransposition process. RESULTS A Minimal Alu RNA Retrotransposition Intermediate To define the minimal sequence requirements for Alu RNA retrotransposition, we adapted a well-established HeLa cell-based assay (Dewannieux et al., 2003; Moran et al., 1996), in which the mobility of tagged Alu elements strictly depends on the coexpression of a functional L1 ORF2 protein (L1ORF2p) that harbors the L1-RT (Figures 1 and S1B). As a reference we used a ‘‘resurrected’’ AluJ element consensus sequence (AJ) (Figure S2 and Table S1) that had previously been shown to retrotranspose with high frequency (Bennett et al., 2008). Surprisingly, we could delete the entire right half monomer from this dimeric Alu RNA without affecting retrotransposition and despite the conserved RNA secondary structure (AJL) (Figure 1). In contrast, a 50 -terminal Alu RNA construct terminating immediately after the Pol III B-box promoter sequence is not sufficient for an effective retrotransposition, indicating additional and important contributions from the Alu RNA ‘‘body’’ (Dewannieux et al., 2003). To delineate which parts of the left half Alu monomer were required for retrotransposition, we truncated the RNA to the minimal Alu folding unit (AJL-H33D) (Figure 1) previously described for SRP RNA (Weichenrieder et al., 1997). This truncation caused a striking reduction in the retrotransposition activity. However, the activity was restored by the re-insertion of the putative B-box sequence for the Pol III promoter (Perez-Stable and Shen, 1986) into the hairpin loop truncating helix H33 (AJL-H33D[B]). Together, these experiments demonstrate that Alu retrotransposition intermediates can be reduced to the minimal SRP Alu folding domain, provided that they contain functional A-box and B-box sequences. The deleted sequences appear to be redundant and dispensable also with regard to potential ‘‘host’’ cell factors that were suspected to interact with these regions (Deininger, 2011). High-Resolution Structure of an Alu RNP in the Closed Conformation To investigate whether the minimal remaining Alu RNA sequence maintained a defined and SRP-like 3D fold, and to rationalize the significance of a defined structure for retrotransposition, we

Please cite this article in press as: Ahl et al., Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.003

A SRP14 SRP9

SRP9

SRP14 loop β1-β2

3’-domain 5’-domain

J12 L31

H2

c-loop

3’

H1

5’

5’-domain

5’ L1

B

D

A 27

5’-domain AJL-H33Δ H31 GAGGCGGG

60 AGGA

CUCUGCC C U A 110

CAG

A GC G CAC

C4

3’-domain

G 25

A 107

G 59

c-loop

L31

3'

G 25

A 57 G5 C 108 G 58

G5

5’-domain

3’-domain

F

G

C 16

G 34

β2 Lys 41

C 35

3’-domain

Ser 48 C 47

A 107 G5

3’-domain

G1

C 37

Leu 46

C 48

C 116

G 15

Val 47

G2

A 50

5’-domain U 24

H0

U 17

L1

β3 SRP9 Pro 2

5’-domain

L2

G7

7.5 Å

L 31

Mg2+

E

Mg2+

H2

U 24

H1

SRP9 Pro 2

SRP9 Lys 41

SRP14 Lys 66

C3

U 26

H1

U 24

H0

A 28 H32

U C G C GA A

L2

3’-domain ~90°

C

C A C U L2 G U U A 40 C G C G H2 C G H0 U A A GGC C A J12 U CCGG GU G C G 5' C G H1 G C 20 C G A C C G G U C U L1 G G

3’

L2

G 49 G 58

H31

G 14

C 108

L 31

I

H SRP14

A 107

J SRP9

G 58

SRP14

5’-domain

Tyr 27 Gly 63 C 108 U 24

C 109 C 110

3’-domain

Mg2+

3’-domain

G5

C4

5’-domain

G 51

Figure 2. High-Resolution Crystal Structure of the Alu RNP in the Closed Conformation (A) Crystallized assembly of SRP9/14 and AJL-H33D RNA (closed conformation). The G5-U24GU motif (orange), the interacting A107C dinucleotide (magenta) in loop L31, and the base pairs between loops L1 and L2 (green) are also shown as sticks (right). Additionally highlighted are the adaptable half of loop L2 (dark gray), the crystallization loop (c-loop, gray), and the magnesium ions (black spheres). (B) Secondary structure of AJL-H33D RNA. (C) The three-way junction with the G5-U24GU motif including the central guanine G25. (D) Primary interface between the Alu RNA 50 - and 30 -domains. Minor groove interactions allow a specific readout of the G5-U24 wobble pair by the sugar edge of adenine A107. (E) Tertiary base pairs between loops L1 and L2 with the adaptable L2 region in gray, including a measure of the narrow H1 major groove (dotted slate). (F) The hinge between the Alu RNA 50 - and 30 -domains and the promotion of chain-reversal by guanine G2 (red). (legend continued on next page)

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Table 1. Data Collection and Refinement Statistics Data Collection Space group

C2

Cell dimensions a, b, c (A˚)

81.48, 56.73, 170.59

a, b, g ( )

90, 93.69, 90

Resolution (A˚)

56.8–2.0 (2.09–2.04)

Rsym (%)

4.8 (65.7)



21.45 (2.03)

Completeness (%)

99.4 (98.1)

Redundancy

3.55 (3.18)

Refinement Resolution (A˚)

56.8–2.0

No. of reflections

49,531

Rwork/Rfree (%)

18.55/22.20

Coordinate error (A˚)a

0.26

No. of atoms All atoms

6,638

Protein

2,557

RNA

3,671

Ligand/ion

29

Water

381

B-factors (A˚2) Overall

35.9

Protein

35.0

RNA

37.1

Ligand/ion

42.0

Water

30.3

Rmsds Bond lengths (A˚)

0.003

Bond angles ( )

0.723

Values in parentheses are for highest resolution shell. Rmsd, root-meansquare deviation. a Maximum likelihood-based coordinate error estimate from phenix.refine.

reconstituted Alu RNPs from RNA and recombinant human SRP9/ 14. Using an RNA that corresponds to the minimal Alu RNA retrotransposition intermediate (AJL-H33D), we obtained Alu RNP crystals that diffracted X-rays to 2.0 A˚ resolution (Figure 2 and Table 1). Structure analysis reveals a highly complex RNA fold that resolves the ambiguities from previous low-resolution studies using human SRP RNA (Weichenrieder et al., 2000). Most important, the Alu RNP can be observed in a fully assembled, closed conformation, in which the helical Alu RNA 30 -domain folds back onto the pseudoknotted Alu RNA 50 -domain (Figure 2A).

The RNA 50 - and 30 -domains interact primarily via shape complementarity, RNA backbone contacts, and numerous water- and ion-mediated contacts that are clearly discernible at high resolution, but surprisingly not via a single base pair interaction (Figures 2A, 2B, 2D, 2G, 2H, and 2J). Further stabilization of the closed conformation comes from nucleotides within the 50 -30 -domain junction and from SRP9/14, which acts like a clamp (Figures 2A, 2B, 2F, 2G, 2I, and 2J). Finally, we observed that the Alu RNA 50 -domain can adapt its structure considerably compared with the previously determined structure of the SRP RNA 50 -domain alone (Figure 3) (Weichenrieder et al., 2000). Plasticity of the 50 -Domain The Alu RNA 50 -domain is characterized by a central three-way junction (J12) of helices H0, H1, and H2 (Figures 2A–2C). The junction is stabilized by a G5-U24GU structural motif that is also recognized by the concave b sheet surface of the SRP9/14 heterodimer (Figure 2A). The G5-U24GU motif consists of a G-U wobble pair (G5-U24) separated from an unusual, three-wayjunction-forming U-turn (U26AA) by an unpaired, cross-strandstacking guanine (G25). Guanine G25 is structurally important also because it stabilizes the junction J12 via water-mediated hydrogen bonds and because it is directly contacted by Lys66 of SRP14 (Figures 2B–2D). The terminal loops of the 50 -domain (L1 and L2) interact via three tertiary base pairs (Figures 2B and 2E). Two of these (G15-C35 and C16-G34) and a ribose-base contact (U17-A33) are also formed in SRP RNA. However, compared with the SRP RNA 50 -domain structure (Weichenrieder et al., 2000), the major groove width at the distal end of helix H1 (U17) narrows from 13.3–7.5 A˚ with a corresponding rotation of loop L1 and its rigidifying U-turn (U13GG) toward the major groove (Figure 3A). This rotation is matched by a hinge motion of helix H2 that shifts its distal end (C35) by 11.0 A˚ to maintain the L1-L2 contacts, indicating an unexpected flexibility of helix H2 and of the terminal loops with respect to the rest of the 50 -domain (Figure 3A). The 30 -terminal half of loop L2 (A36CUUU compared with U35ACUC in SRP RNA) does not seem to play a specific role in the assembly. It is squeezed into a narrow hairpin ‘‘tongue’’ and appears to be structurally adaptable (Figure 3A). Specificity of the 50 -30 -Domain Interface The Alu RNA 30 -domain is stabilized in the closed conformation by a primary interface that forms between the G5-U24GU structural motif in the 50 -domain and the A107C dinucleotide in loop L31 of the 30 -domain (Figures 2B, 2D, 2G, 2H, and 2J). The nucleotides of this internal, asymmetric loop engage in Watson-Crick (G58C108) and tandem GA/AG pairs (G59-A107 and A60-G106). This results in a defined, helical arrangement of loop L31 with a minor

(G) Stabilization of the closed Alu RNP conformation by SRP9 (salmon) and recognition of the L31 minor groove. In (C) to (G), magnesium ions (gray) and waters (purple) are shown as spheres, and tertiary hydrogen bonds are dotted black. (H) Electron density from a simulated-annealing composite omit 2Fo-Fc map, contoured at 1.0 s. (I) Protein-RNA interface. The interface (1,820 A˚2) extends across the Alu RNA 50 -domain (orange, 1,400 A˚2) and 30 -domain (yellow, 420 A˚2), with contributions from SRP9 (700 A˚2) and SRP14 (1,120 A˚2). All interfacial waters (purple) and ligands (black sticks, putative acetate ion and PEG fragment) are shown. (J) Alu RNA 30 -domain interface. The interface (1,590 A˚2) extends across the Alu RNA 50 -domain (orange, 1,150 A˚2) and SRP9/14 (yellow, 440 A˚2) and includes waters (purple) and a magnesium ion (black).

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groove that is perfectly complementary to the minor groove of the opposing G5-U24 wobble pair from the G5-U24GU motif. A magnesium ion, specifically coordinated in the major groove by G58 and G59, further stabilizes the RNA backbone where adenine A57 points toward the solvent (Figure 2D). The structure shows that the A107C dinucleotide cannot be substituted without disrupting the domain interface. On C108, the exocyclic amino group contacts the A107 phosphate to stabilize the RNA backbone in the S-shaped conformation necessary to interact with the 50 -domain. On A107, the phosphate and the sugar edge are read out specifically by the G5-U24 wobble pair, including a U24A107 ribose zipper and discrimination against a guanine, because there is insufficient space for the exocyclic amino group. Additional stabilization of the interface is provided by a magnesium ion bridging the A107 and C108 phosphates to the G5 ribose, from a second ribose zipper (C23-U61), and from a series of water-mediated contacts (Figures 2D and 2H). RNA Strand Reversal at the 50 -30 -Domain Junction At the hinge between the two Alu RNA domains, RNA strand reversal and back-folding of the 30 -domain is guided by guanine G2 from helix H0, stabilizing the closed conformation (Figures 2B and 2F). Guanine G2 uses its sugar edge and a ribose zipper to fix the backbone of G49 and A50 at the beginning of helix H31. Further stabilization comes from G1 (absent in SRP RNA), which base pairs with C48 (in helix H0) and forms another ribose zipper with C116 (in helix H31). Stabilization via the SRP9 Protein In mammalian SRP RNA, the closed Alu RNA conformation requires the stabilization by the SRP9/14 heterodimer (Weichenrieder et al., 2001; Wild et al., 2002). SRP9/14 acts like a clamp on the two RNA domains (Figures 2A, 2I, and 2J), and consequently we observed multiple interactions of SRP9 with the Alu RNA 30 -domain (Figures 2B, 2D, and 2G). These include SRP9Lys41, which bridges the G25 phosphate and the A107 adenine base via a water molecule. Furthermore, the main-chain carbonyl oxygens of SRP9-Asp45 and SRP9-Leu46 in the SRP9 loop L(b2-b3) read out the sugar-edge of G58 in the L31 minor groove (Figure 2G). Interestingly, the SRP9 N-terminal nitrogen (on Pro2, fixed to the b3-strand) makes an RNA phosphate backbone contact to A60 (Figures 2D and 2G), similar to the N-terminal contact of SRP14 with the phosphate of C30 (see also Weichenrieder et al., 2000). Interactions with the Ribosomal Elongation FactorBinding Site Single-particle reconstruction from cryo-EM data has revealed that the Alu domain of the mammalian SRP binds to the elongation factor-binding site at the interface between the small and large ribosomal subunits, where it interferes with ongoing elongation (Halic et al., 2004). Because ribosome binding has also been suggested to play a role in Alu retrotransposition (Boeke, 1997), we used rigid-body fitting to dock our crystal structure of a fully assembled Alu RNP into the deposited cryo-EM electron density (Figures 3B and S3). For this purpose, we first made use of an improved 7.4 A˚ resolution map of the mammalian SRP on wheat-germ ribosomes (Figure 3B) (Halic et al., 2006)

that showed much clearer density for SRP9/14 than the originally published 12 A˚ resolution map (Halic et al., 2004). Second, we directly superimposed the recently published ribosome-SRP complex from rabbit reticulocytes for interspecies comparison (Figure S3) (Voorhees and Hegde, 2015). We observed that the conformational difference in the AluJ RNA 50 -domain is important, because it permits a better fit of the 50 -domain and especially of the helix H2-loop L2 (H2-L2) hairpin structure into the cryo-EM density than the SRP-derived 50 -domain structure (Figure 3B). Furthermore, the cryo-EM density also supports the crystallized position and orientation of the Alu RNA 30 -domain (Figure 3B). In conclusion, the ribosomal placement of the Alu RNP is defined by three separate interfaces and consequently, in the context of the SRP, may not need the SRP S domain for orientation (Figures 3B and S3). These interfaces are (1) between the L1-L2 base-pairing loops and the flexible P stalk of the large ribosomal subunit; (2) between the elongation arrest-mediating surfaces of SRP9/14 (Lakkaraju et al., 2008; Mary et al., 2010) and the RNA at the shoulder of the small ribosomal subunit; and (3) between the minor groove surface of Alu RNA helix H1 and the highly conserved (Voorhees and Ramakrishnan, 2013) sarcin-ricin loop of large rRNA (Figure S3). The cryo-EM reconstruction was obtained from ribosomenascent chain complexes that were arrested in translation and that stabilized the Alu domain of the SRP in the elongation factor-binding site (Halic et al., 2004; Voorhees and Hegde, 2015). Notably, the resulting position of the Alu domain is incompatible with the simultaneous presence of elongation factors. In a positional overlay with the human eEF2-ribosome complex (Anger et al., 2013), the Alu RNA 50 -domain corresponds to the G domain of eEF2 (eukaryotic elongation factor 2), the Alu RNA 30 -domain to the G0 domain, and SRP9/14 to the b-barrel domain II (Figure 3C). Accordingly, the ribosomal contact surfaces are also similar, including the sarcin-ricin loop, which in the elongation factors faces the highly conserved nucleotide-binding pocket of the G domain (Gao et al., 2009). In contrast to the elongation factors, however, Alu RNPs would tolerate the presence of an A-site tRNA, suggesting several states of ribosomal arrest that could be targeted by an Alu RNP (Figure S4). Conservation of SRP Alu RNA Structure and Interactions In order to understand which parts of the Alu RNP are relevant for RNA folding and for potential ribosomal interactions, we performed a detailed structural comparison of SRP Alu RNA structures across all three taxonomic kingdoms. This analysis was facilitated by recent SRP Alu RNA crystal structures from an archaeon, Pyrococcus horikoshii (Bousset et al., 2014), and from a bacterium, Bacillus subtilis (Kempf et al., 2014), which could be structurally superimposed onto the present, eukaryotic Alu RNP (Figure 4). In all three structures (Figures 4A–4C), the Alu RNA 50 -domain interacts with the Alu RNA 30 -domain via an interface formed between junction J12 and loop L31, as anticipated from secondary structure comparisons (Wild et al., 2002). Strikingly, the readout of the invariant G-U wobble pair in junction J12 by the adenine from loop L31 is identical in all three cases and assisted by a conserved ribose zipper between U61 and C23 that further fixes Molecular Cell 60, 1–13, December 3, 2015 ª2015 Elsevier Inc. 5

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A

3’ SRP14

5’-domain H2

3’

L2

5’

Figure 3. Alu RNP Interactions in the Ribosomal Translation Elongation Factor-Binding Site

5’-domain

5’

L2 H2

13.3 Å

7.5 Å

L1

L1 H1

H1 SRP9

3’-domain 3’

H2 H2

SRP14 5’

L2 L2

11 Å

5’

7.5 Å

L1 H1

~90°

SRP9

3’-domain

3’-domain

(A) Structural comparison of the minimized Alu RNP (AJL-H33D, 2.0 A˚ resolution, RNA in slate and cyan) to the 50 -domain of SRP RNA (PDB: 1E8O) (Weichenrieder et al., 2000) (3.2 A˚ resolution, RNA in red). Top: side-by-side views, including a measure of the H1 major groove (dotted lines). Bottom: superposition via SRP9/14, illustrating the shift of helix H2 and the reorientation of terminal loops L1 and L2, with the adaptable region of loop L2 in gray. (B) Placement of the Alu RNP crystal structures and of a model for the wheat-germ ribosome (Armache et al., 2010) into the 7.4 A˚ cryo-EM envelope of the wheat-germ ribosome occupied by the canine SRP (Halic et al., 2006). The large (60S) and small (40S) ribosomal subunits and the sarcin-ricin loop (SRL) are labeled, with RNA in purple and protein in gray. The closure of the helix H1 major groove is matched by a hinge motion of helix H2 that leads to a better fit of the 50 -domain into the cryo-EM envelope and places the adaptable part of loop L2 in a different orientation with respect to the N-terminal domain of the L11 protein. Left: side view. Right: top view. (C) Positional overlay of the Alu RNP with domains I (G and G0 ) and II of eEF2, obtained from their ribosomal positions as described in Supplemental Experimental Procedures. See also Figures S3 and S4.

B small subunit (40S)

large subunit (60 S)

h15

Prokaryotes apparently lack SRP9/14 homologs that could stabilize the closed h15 H43 conformation. Instead, the SRP RNAs H43 from P. horikoshii and B. subtilis form an h5 H44 additional helix (Hterm) between their H44 h14 extended 50 - and 30 -sequences. This helix L11 folds into a defined three-way junction P0 P0 with RNA helices H0 and H31 that seems SRL S24 to fix their orientation without the need for h8 P-stalk additional, protein-mediated stabilization (Figures 4B and 4C). C eEF2 The two prokaryotic structures also reveal domain II eEF2 eEF2 extended loop L2 sequences that allow for G domain II additional Watson-Crick base pairs with loop L1. This results in surprisingly similar structures considering their evolutionary distance (Figures 4B and 4C). In contrast, eEF2 eEF2 G loop L2 and the L1-L2 loop-loop interaction domain I are much less well conserved in eukaryotic eEF2 SRP RNAs, where loop L2 is often severely G’ eEF2 reduced. In fungi such as Saccharomyces G’ cerevisiae, the only remaining elements of the 50 -domain are the G-UGU motif with the G-U wobble pair and the adjacent base pairs of helix H1 (Figure 4D). Consequently, the helix H1 minor groove represents one of the RNA helix H1. Furthermore, although there is no sequence equivalent for G25 in the bacterial structure, B. subtilis A37 takes the most conserved Alu RNA surfaces. On the ribosome, it faces a structurally equivalent role in stacking on the G-U wobble pair the surface of the highly conserved sarcin-ricin loop that otherwise contacts the guanine nucleotide binding site of the (Figure 4C). SRL

L11

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A

C

G

A C

C AU A U GU C C G H1 C A C U C G

U U U

H0

J12

C

G

U A U

G C C G G

AGGA

C UA

C

G 25

3’-domain

5'

C 23

A 107

U 61

J12

L31

C 108

H31

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H32 5’-domain

P. horikoshii

H0

U

H1

H0/H2 3'

U 24

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J12

L2 L31

H31 31

AG C C C G AGG

GGC

G 17 U 35

H32

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C CUU UG C G G G C U C C CAC G 123 C C H U term C

5' 3'

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L1

3'

GGC C GA

C

C

G5

H32

UCGC

C UCUGC C C AGCG CAG U 107 07 A

C C G G AU G C G 17 G G G G G G G G U

J12

C

GAGGC GGG

A L2

C CA

A A G G H2 C C A A U GU C C C H1 C U G C G G

L2

L31

H31

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G

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H. sapiens (Alu) lu)

C CGG G G 5 5' G C G C G G U G L1 C

C

G

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G G G A

H2 C

B

L2

A

H0/H2

H1

G 36

C 34

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3'

G 80

J12

J12

Hterm

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L1

L31

5'

L2

C 124

H31

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H32 5’-domain

A B. subtilis C G G C A L31 G H31 C G H0 C G A H32 A GG UU GC CGA AU C C C UUC U

L2

G 16 U 34

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U G U C C C H1 G U G G C G

J12

C G AAU G C G 16 G G U G G A C G C G G G U A L1

U G C A C G G U C

G G A AG A

CAG 93

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5’-domain H1

Hterm

G

J12

Hterm

A C C C GU

G G G A

A U GU C C G U G C G C G G

A C U AG G U GU U C G C G G G C G

U

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H. sapiens (SRP) RP)

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S. cerevisiae UGGA CAG

UGGA ACUU

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5'

Figure 4. Ancient Origin and Structural Conservation of the Alu Domain in SRP RNAs from All Three Taxonomic Kingdoms (A) Human Alu RNA. Left: secondary structure with structural elements in distinct colors. The invariant G-U wobble pair in junction J12 and its interacting adenine from L31 are boxed and numbered. The structurally conserved part of helix H1 is in orange, the remainder of the junction J12 is in yellow, and the interacting loop L31 is in pink, with important nucleotides highlighted in magenta. Dashes mark Watson-Crick base pairs, dots mark wobble pairs, and crosses mark non-canonical pairs (thinner in case of a single hydrogen bond). Nucleotides engaged in tertiary loop L1-loop L2 Watson-Crick base pairs are circled, other structurally important and conserved nucleotides are outlined in black. U-turns are indicated by an arc, and helix stacking by an elbow bar. Middle: crystal structure with nucleotides guiding Alu RNA assembly shown as cylinders or sticks. Right: conserved interface between the Alu RNA 50 - and 30 -domains. Structurally conserved hydrogen bonds are shown as dotted lines including the readout of the G5-U24 wobble pair by A107, the stabilization of the L31 backbone by C108 and the ribose zipper between C23 and U61. (B) Archaeal Alu RNA from P. horikoshii (PDB: 4UYK) (Bousset et al., 2014). (C) Bacterial Alu RNA from B. subtilis (PDB: 4WFL) (Kempf et al., 2014). (D) Eukaryotic SRP Alu RNA secondary structures, illustrating the variability of the H2-L2 hairpin compared with the conservation of helix H1 and of the 50 -30 -domain interface. Sequences are taken from the SRP database (Rosenblad et al., 2009). (E) Superposition of the Alu RNA structures including the orientation with respect to the sarcin-ricin loop (SRL) in the ribosomal translation elongation factor-binding site. The GDP nucleotide illustrates the position of the guanine nucleotide binding site of EFG from a crystal structure with the bacterial ribosome (Gao et al., 2009).

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elongation factors (Figure 4E). This suggests an evolutionary conserved ribosomal binding mode of the Alu RNA. Alu RNP Structure in Retrotransposition The analysis of the various crystal structures demonstrates that the Alu RNA 50 -30 -domain interaction, mediated by loop L31, is a highly and precisely conserved feature in SRP RNAs. In contrast, the interactions and precise orientations of loops L1 and L2 are less well preserved, in particular among eukaryotic SRP RNAs. Therefore, we investigated the relevance and functional role of the respective Alu RNA structural elements (L1, L2, L31) in retrotransposition, together with a previously described mutation of junction J12 (G25C) (Bennett et al., 2008; Chang et al., 1997) in the 50 -domain of an AluY element (Figure 5). Importantly, we not only compared the number of successful retrotransposition events for the various RNA mutants, but we also used northern blots to normalize retrotransposition to the respective Alu RNA levels in the cells (Figures 5A–5E, left versus right). In this way we corrected for the non-structural effects of the mutations, such as mutations and deletions in loop L1 that partially affected the putative A-box of the RNA Pol III promoter (Figure 5B) (PerezStable and Shen, 1986). Furthermore, all of the experiments were performed in the context of a monomeric Alu retrotransposition intermediate (AJL) in order to exclude the possibility of compensatory effects from the intact right half monomers. First, we demonstrated that the regularization of helix H33 (i.e., its conversion into a fully base-paired RNA helix [AJLH33p]), did not change the ability to retrotranspose, similar to the deletion of the entire helix H33 (Figure 5A). Second, we tested the importance of the L1-L2 loop-loop interaction by either mutating (AJL-L1m, AJL-L2m) or deleting (AJL-L1D, AJL-L2D) the respective nucleotides. We found that an interaction of the two loops is apparently not required for retrotransposition and that manipulations of loop L2 are tolerated much better than manipulations of loop L1. This observation is consistent with a direct structural role of loop L1 and/or of the adjacent helix H1 in retrotransposition (Figure 5B). Third, variations of loop L31 that interfere with the 50 -30 domain interaction clearly reduce the ability to retrotranspose. Such manipulations either disrupted the internal stabilization of loop L31 (AJL-L31m) or converted it into a regular RNA helix (AJL-L31p). In contrast, an AluY-like mutation of loop L31 (AJL-L31Y) that preserves the 50 -30 -domain interface had only a relatively modest effect (Figure 5C). Taken together, these experiments are consistent with the notion that the 30 -domain contributes to an efficient retrotransposition and in particular that its orientation within the RNP is functionally significant. A folded Alu RNA 50 -domain, recognized by SRP9/14 like the SRP RNA 50 -domain in Figures 3A and 3B, is not sufficient for full activity. Additional support for the importance of the closed RNA conformation in retrotransposition comes from mutations of junction J12 in the 50 -domain and from comparisons of SRP9/14 affinity. For this purpose, we calculated differential Alu RNA binding energies (DDG) from in vitro RNA competition experiments, as previously described (Bennett et al., 2008; Weichenrieder et al., 1997, 2001). Surprisingly, we found that the mutation of junction J12 (G25C) in AJL (AJL-J12m) did not reduce retrotransposition detectably (Figure 5D), although this mutation reduced SRP9/14 affinity by 8 Molecular Cell 60, 1–13, December 3, 2015 ª2015 Elsevier Inc.

more than 6-fold (AJL-J12m, DDG = 1.1 kcal/mol) and despite the observation that this mutation almost abolished retrotransposition in the context of a modern AluY left monomer (AJL-YJ12m versus AJL-Y) (Figure 5E). In the context of the AluJ monomer, the mutation of junction J12 only abolished retrotransposition after converting loop L31 into a regular helix (AJL-L31p-J12m) (Figure 5D), a modification that, on its own, already reduced SRP9/14 affinity by more than 30-fold (AJLL31p, DDG = 2.1 kcal/mol). We therefore conclude that a limited destabilization of the Alu RNP structure is tolerated but that the loss of SRP9/14 interaction (DDG > 3.5 kcal/mol) clearly abolishes retrotransposition even if the Alu RNA 50 - and 30 -domains remain largely intact. However, there is no simple correlation between SRP9/14 affinity and retrotransposition activity (Figure 5F). This suggests that structural features of the Alu RNP must play a crucial role. Mutations are tolerated better if the overall shape of the complex remains intact (AJL-J12m), and retrotransposition decreases when the closed conformation begins to open up (AJL-L31p). Furthermore, AYL-specific mutations in the Alu RNA 30 -domain apparently have a stimulatory effect, although this region is not involved in SRP9/14 interactions and can be deleted from AJL (AJL-Y versus AJL-L31Y) (Figure 5E). We suggest that these mutations either facilitate the directional unfolding of the Alu RNA when it is due to be reverse transcribed by the L1ORF2p or that they facilitate another mechanistic step that is rate limiting in the retrotransposition cycle. DISCUSSION Ancient Origin of the Alu RNP Structure We determined the first high-resolution structure of a human Alu RNP and demonstrated the necessity of this assembly for the success of the Alu retrotransposon as the primary human genomic parasite. The structure is composed of SRP9/14 and of the RNA sequences that align to the minimal Alu RNA folding domain of the human SRP RNA. The RNA consists of two structural domains, a pseudoknotted 50 -domain that interacts with a helical 30 -domain by shape complementarity. The RNA domain interface is conserved in archaeal and bacterial SRP RNAs, indicating an ancient origin of the Alu RNA structure. Apart from the interface, the most highly conserved structural element in Alu RNA is the helix H1. In the interaction with the ribosome, it faces the elongation factor-binding surface of the sarcinricin loop, one of the most highly conserved elements of the large ribosomal subunit. Therefore, not only the Alu RNA structure per se but also the interaction with the ribosome is likely to be of ancient origin, perhaps even dating from the RNA world. An ancient Alu RNA could also have assumed a different function, such as the role of a primordial translation elongation factor rather than that of an interfering obstacle for translation elongation. This scenario may well have altered only with the advent of more efficient, protein-based replacements in the form of eEF2 and eEF1A (EFG and EFTu in bacteria) (Figure S4A). Requirements for Retrotransposition The SRP Alu RNA sequences of higher eukaryotes require SRP9/14 to fold into the closed conformation (Weichenrieder

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A

B

C

D

E

F

(legend on next page)

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Please cite this article in press as: Ahl et al., Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.003

A

arrested ribosome ΙI

5’

ΙI

L1 ORF1p

stalling ribosome Alu-int

3’AAAAA

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mRNA

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nascent chain

B

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Ι nascent L1 ORF2p

L1 RT

EN

Alu-int ΙII nascent L1 ORF2p

L1 RT

EN

A A A A A 3’

3’AAAAA

Figure 6. Model for Alu RNP Function in Retrotransposition (A) SRP-mediated elongation arrest (Nyathi et al., 2013). On translating ribosomes, nascent chain recognition by the S domain (I) lets the Alu domain out-compete elongation factors (II), causing elongation arrest (III). (B) Proposed mechanism for ‘‘L1 cis-preference by elongation arrest’’ and its exploitation by Alu retrotransposition intermediates (Alu-int). Left: on rapidly translating ribosomes (I), Alu RNAs are out-competed by elongation factors (II). Middle: Ribosomal stalling favors L1 cis-preference, but stalling (I) also creates a preferential binding state for Alu RNAs (II). Right: enhanced stalling (III, elongation arrest) by Alu RNAs helps them overcome L1 cis-preference and hijack L1ORF2p via their oligo(A) tail. EN, endonuclease; RT, reverse transcriptase. Protein and RNA shapes are in scale using structures from the following PDB entries: 5AOX for the Alu RNP, 1RY1 for the SRP model (Halic et al., 2004), 3J5Z, 3J60, 3J61, 3J62 for the ribosome (Armache et al., 2010), 1VYB for the L1ORF2pendonuclease (Weichenrieder et al., 2004), and 2YKO for the L1ORF1p trimer (Khazina et al., 2011).

et al., 2000, 2001). Furthermore, SRP9/14 is required to recognize and stabilize the ribosome in the arrested state (Halic et al., 2004; Lakkaraju et al., 2008; Mary et al., 2010; Nyathi et al., 2013; Voorhees and Hegde, 2015). It has remained unclear, however, to what degree the Alu RNP structure was relevant also for Alu retrotransposition. With our structure-based mutational analysis, we confirm that a loss of SRP9/14 interaction abolishes Alu retrotransposition (Bennett et al., 2008). However, the correlation between SRP9/14 affinity and retrotransposition is complicated, and conformational differences between the Alu RNA variants are also critical, in particular if they affect the ability to maintain the closed conformation. Importantly, we find that the Alu RNA sequences and structures that vary most in the evolution of SRP RNA are also most tolerant toward mutations in Alu retrotransposition. This is particularly relevant for the H2-L2 hairpin structure. This hairpin is frequently reduced or deleted in eukaryotic SRPs, probably because SRP9/14 has taken over some of the ribosome binding functions. Our structural analysis demonstrates the plasticity of the H2-L2 hairpin and that Alu retrotransposition works well even if loop L2 is deleted. In contrast, the H1-L1 hairpin, which likely positions the Alu RNP with respect to the sarcin-ricin loop, is much more conserved in SRP evolution and does not tolerate mutations well in the context of retrotransposition. Together, the data presented in this study support a crucial role of Alu RNP structure in retrotransposition, possibly at

several distinct steps in the retrotransposition cycle. As in the SRP, the Alu RNP structure will protect the RNA from nuclease attack and likely facilitates nuclear export. However, ribosome binding is probably the most demanding function of the Alu RNP from a structural point of view and therefore imposes most of the long-term evolutionary constraints. Apart from the present Alu RNP structure, Alu retrotransposition intermediates also require a poly(A) sequence (Dewannieux et al., 2003). In the publication accompanying the present work in this issue of Molecular Cell, Doucet et al. (2015) now demonstrate for LINE-1 RNA that the poly(A) sequence serves to recruit the L1 ORF2 protein. They also show that, upon artificial removal of the poly(A) tail from L1 RNA, Alu RNA retrotransposes with increased efficiency, in contrast to other poly(A) containing RNA substrates. This observation points at a close association of L1 and Alu RNA at the site of L1ORF2p production and further corroborates the ribosome-binding hypothesis. Ribosome Stalling The ability of Alu retrotransposition intermediates to form an SRP-like Alu RNP structure (Figure 2), the fit of this structure into the ribosomal translation elongation factor-binding site (Figure 3B), and the functional requirement of the crystallized, closed Alu RNA conformation (Figures 1 and 5) strongly support a role of ribosome-binding in Alu retrotransposition that was preserved from the parental SRP despite the absence of an SRP S-like

Figure 5. Structural Requirements for Retrotransposition Retrotransposition activity of AluJ RNA structural variants is shown before normalization to RNA levels (left, dark blue) and after RNA normalization (right, light blue). RNA levels used for normalization are plotted in red (left) and differential Alu RNA binding energies (DDG) of SRP9/14 are plotted in orange (right). Data are represented as mean ± SEM (n = 3). RNA variants with AluY-like mutations are labeled in gray. Mutations are illustrated on Alu RNA schemes with further details in Figure S5 and Table S1. (A) Dispensability of the right half Alu monomer and of the extended 30 -stem. (B) Dependence of retrotransposition on the H1-L1 hairpin structure compared with the relevance of the H2-L2 hairpin structure or the L1-L2 loop-loop interaction. (C) Importance of the loop L31 structure. (D) Conditional sensitivity of retrotransposition to the G25C 50 -domain mutation in comparison with the SRP9/14 binding capacity. (E) Enhanced retrotransposition of AluY RNA and increased sensitivity to the G25C 50 -domain mutation. (F) Retrotransposition activity plotted as a function of differential SRP9/14 affinity (DDG).

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domain. Furthermore, ribosome-binding provides a powerful rationale for the preferential access of Alu RNAs to nascent L1ORF2p, compared with other poly(A)-containing RNAs in the cell (Boeke, 1997; Doucet et al., 2015). It remains unclear, however, how Alu retrotransposition intermediates selectively identify ribosomes that translate L1ORF2p and how they avoid to prematurely arrest L1ORF2p elongation before the protein is of sufficient length to function in poly(A) RNA binding. Thus, possible variations in the kinetics of L1ORF2p translation elongation could be a crucial factor in L1 and Alu retrotransposition, and translational stalling (Lu and Deutsch, 2008; Voorhees and Hegde, 2015; Wilson and Beckmann, 2011) could be central to the L1 cis-preference. A slow progression of the large ribosomal subunit on, for example, the lysine-rich sequences in the C-terminal region of L1ORF2p would prolong the half-life of the nascent L1ORF2p-ribosome complex and increase the probability for the already folded parts of the protein to trap the poly(A) tail of the encoding L1 mRNA. This mechanism could be called ‘‘cis-preference by elongation arrest’’ (Figure 6). At the same time, however, the translationally impeded ribosomes would become much better targets for Alu RNPs than rapidly elongating ribosomes, because Alu RNPs preferentially bind and stabilize the arrested state. This model is in agreement with the view that also in the context of the SRP, the Alu domain can bind independently of a displayed signal sequence and serves to read out rather than to induce an elongation arrest (Voorhees and Hegde, 2015). Ribosome stalling may thus provide a common mechanistic basis, both for the L1 cis-preference and for the success of the Alu element to exploit the L1 retrotransposition machinery. EXPERIMENTAL PROCEDURES Sample Preparation Alu RNAs were generated by large-scale in vitro run-off transcription (using T7 RNA polymerase and linearized plasmid templates), followed by gel purification as previously described (Weichenrieder et al., 1997). The SRP9 and SRP14 proteins were expressed separately in the Escherichia coli strain BL21 (DE3) Rosetta II and purified as a heterodimer from an MBPTrap HP column (5 ml; GE Healthcare), followed by tag-removal, heparin affinity, and size-exclusion chromatography steps. See Supplemental Experimental Procedures for further details. Crystallization To reconstitute Alu RNPs for crystallization, RNA was first re-annealed (10 min at 65 C, followed by slow cooling over 3–4 hr in 10 mM Na-HEPES [pH 7.5], 10 mM MgCl2, 100 mM NaCl) and mixed with a 1.15-fold molar excess of SRP9/14, resulting in a complex solution containing 100 mM RNA, 115 mM SRP9/14, 8.3 mM Na-HEPES [pH 7.5], 0.6 mM DTT, 10 mM MgCl2, 15 mM (NH4)2SO4, and 123 mM NaCl. RNPs were equilibrated for 15 min at room temperature before setting up crystallization trials. Crystals were obtained by hanging drop vapor diffusion at 18 C, mixing 1 ml of reconstituted RNP complex with 1 ml of reservoir solution (0.1 M Na-MES [pH 6.4], 30% PEG 400) over a 500 ml reservoir. Crystals were snap-frozen in liquid nitrogen after adding 2 ml of reservoir solution to the crystallization drop for cryoprotection. Data Collection and Structure Determination Diffraction data extending to a resolution of 2.0 A˚ were collected at a wavelength of 1.000 A˚ on beamline PXIII of the Swiss Light Source. The structure was solved by molecular replacement, using a complex of SRP9/14 from Protein Data Bank (PDB) accession number 1E8O (Weichenrieder et al.,

2000) as a search model. The final model contains two copies of the complex per asymmetric unit. Residues P41-G48 from the SRP14 loop L(b2-b3) are disordered and missing from the model. Nucleotides C68 and A99 in the loop truncating the Alu RNA 30 -domain are poorly defined in the density and likely adopt alternative conformations. For illustrations, these nucleotides were modeled stereochemically. See Supplemental Experimental Procedures for further details and structural superpositions. Alu Retrotransposition The principle of the retrotransposition assay, in which retrotransposition from a source plasmid into the genome of HeLa cells results in resistance against G418, has been described previously (Dewannieux et al., 2003; Moran et al., 1996). To score Alu retrotransposition with respect to RNA transcript levels, cells were split evenly into different culture dishes 3 days after transfection. The next day, one dish was used to start the G418 selection and another dish was used to extract RNA for northern blot analysis. Sixteen days after transfection, G418-resistant colonies were stained, counted, and normalized to the number obtained for the Alu AJL construct. Experiments were conducted three times each, including northern blot analysis. See Supplemental Experimental Procedures for further details, including northern blot analysis. ACCESSION NUMBERS The accession number for the atomic coordinates and structure factors reported in this paper is PDB: 5AOX. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.molcel.2015.10.003. AUTHOR CONTRIBUTIONS V.A. designed experiments, did all the experiments, analyzed the data, and drafted the paper. H.K. provided RNA and protein plasmids and performed initial experiments. S.S. helped in structure and competition data analysis. O.W. conceived the project, designed experiments, analyzed the data, and finalized the manuscript together with V.A. All authors discussed the results and commented on the manuscript. ACKNOWLEDGMENTS We are grateful to Elisa Izaurralde and department members for comments on the manuscript. We thank Regina Bu¨ttner for excellent technical assistance, Belinda Loh for help with northern blot analysis, and the staff of the Swiss Light Source for assistance during data collection. Furthermore, we thank Jose´-Luis Garcı´a-Pe´rez and John Moran for plasmids to start the retrotransposition assay, sequence information, and advice. This work was supported by the Max Planck Society. Received: July 15, 2015 Revised: September 14, 2015 Accepted: October 1, 2015 Published: November 12, 2015 REFERENCES Ade, C., Roy-Engel, A.M., and Deininger, P.L. (2013). Alu elements: an intrinsic source of human genome instability. Curr. Opin. Virol. 3, 639–645. Anger, A.M., Armache, J.P., Berninghausen, O., Habeck, M., Subklewe, M., Wilson, D.N., and Beckmann, R. (2013). Structures of the human and Drosophila 80S ribosome. Nature 497, 80–85. Armache, J.P., Jarasch, A., Anger, A.M., Villa, E., Becker, T., Bhushan, S., Jossinet, F., Habeck, M., Dindar, G., Franckenberg, S., et al. (2010).

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Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution. Proc. Natl. Acad. Sci. U S A 107, 19748–19753. Baillie, J.K., Barnett, M.W., Upton, K.R., Gerhardt, D.J., Richmond, T.A., De Sapio, F., Brennan, P.M., Rizzu, P., Smith, S., Fell, M., et al. (2011). Somatic retrotransposition alters the genetic landscape of the human brain. Nature 479, 534–537. Batzer, M.A., and Deininger, P.L. (2002). Alu repeats and human genomic diversity. Nat. Rev. Genet. 3, 370–379. Bennett, E.A., Keller, H., Mills, R.E., Schmidt, S., Moran, J.V., Weichenrieder, O., and Devine, S.E. (2008). Active Alu retrotransposons in the human genome. Genome Res. 18, 1875–1883. Boeke, J.D. (1997). LINEs and Alus–the polyA connection. Nat. Genet. 16, 6–7. Bousset, L., Mary, C., Brooks, M.A., Scherrer, A., Strub, K., and Cusack, S. (2014). Crystal structure of a signal recognition particle Alu domain in the elongation arrest conformation. RNA 20, 1955–1962.

Kriegs, J.O., Churakov, G., Jurka, J., Brosius, J., and Schmitz, J. (2007). Evolutionary history of 7SL RNA-derived SINEs in Supraprimates. Trends Genet. 23, 158–161. Lakkaraju, A.K., Mary, C., Scherrer, A., Johnson, A.E., and Strub, K. (2008). SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites. Cell 133, 440–451. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., et al.; International Human Genome Sequencing Consortium (2001). Initial sequencing and analysis of the human genome. Nature 409, 860–921. Levin, H.L., and Moran, J.V. (2011). Dynamic interactions between transposable elements and their hosts. Nat. Rev. Genet. 12, 615–627. Lu, J., and Deutsch, C. (2008). Electrostatics in the ribosomal tunnel modulate chain elongation rates. J. Mol. Biol. 384, 73–86.

Burns, K.H., and Boeke, J.D. (2012). Human transposon tectonics. Cell 149, 740–752.

Luan, D.D., Korman, M.H., Jakubczak, J.L., and Eickbush, T.H. (1993). Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605.

Chang, D.Y., Newitt, J.A., Hsu, K., Bernstein, H.D., and Maraia, R.J. (1997). A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14. Nucleic Acids Res. 25, 1117–1122.

Macia, A., Mun˜oz-Lopez, M., Cortes, J.L., Hastings, R.K., Morell, S., LucenaAguilar, G., Marchal, J.A., Badge, R.M., and Garcia-Perez, J.L. (2011). Epigenetic control of retrotransposon expression in human embryonic stem cells. Mol. Cell. Biol. 31, 300–316.

Cost, G.J., Feng, Q., Jacquier, A., and Boeke, J.D. (2002). Human L1 element target-primed reverse transcription in vitro. EMBO J. 21, 5899–5910. de Koning, A.P., Gu, W., Castoe, T.A., Batzer, M.A., and Pollock, D.D. (2011). Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet. 7, e1002384. Deininger, P. (2011). Alu elements: know the SINEs. Genome Biol. 12, 236. Deininger, P.L., Jolly, D.J., Rubin, C.M., Friedmann, T., and Schmid, C.W. (1981). Base sequence studies of 300 nucleotide renatured repeated human DNA clones. J. Mol. Biol. 151, 17–33. Dewannieux, M., Esnault, C., and Heidmann, T. (2003). LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet. 35, 41–48. Doucet, A.J., Wilusz, J.E., Miyoshi, T., Liu, Y., and Moran, J.V. (2015). A 30 poly(A) sequence is required for LINE-1 retrotransposition. Mol. Cell 60, Published online November 12, 2015. http://dx.doi.org/10.1016/j.molcel. 2015.10.012.

Mary, C., Scherrer, A., Huck, L., Lakkaraju, A.K., Thomas, Y., Johnson, A.E., and Strub, K. (2010). Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein. RNA 16, 969–979. Moran, J.V., Holmes, S.E., Naas, T.P., DeBerardinis, R.J., Boeke, J.D., and Kazazian, H.H., Jr. (1996). High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927. Nyathi, Y., Wilkinson, B.M., and Pool, M.R. (2013). Co-translational targeting and translocation of proteins to the endoplasmic reticulum. Biochim. Biophys. Acta 1833, 2392–2402. Okada, N., Hamada, M., Ogiwara, I., and Ohshima, K. (1997). SINEs and LINEs share common 30 sequences: a review. Gene 205, 229–243. Perez-Stable, C., and Shen, C.K. (1986). Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat. Mol. Cell. Biol. 6, 2041–2052.

Gao, Y.G., Selmer, M., Dunham, C.M., Weixlbaumer, A., Kelley, A.C., and Ramakrishnan, V. (2009). The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699.

Rosenblad, M.A., Larsen, N., Samuelsson, T., and Zwieb, C. (2009). Kinship in the SRP RNA family. RNA Biol. 6, 508–516.

Halic, M., Becker, T., Pool, M.R., Spahn, C.M., Grassucci, R.A., Frank, J., and Beckmann, R. (2004). Structure of the signal recognition particle interacting with the elongation-arrested ribosome. Nature 427, 808–814.

Shukla, R., Upton, K.R., Mun˜oz-Lopez, M., Gerhardt, D.J., Fisher, M.E., Nguyen, T., Brennan, P.M., Baillie, J.K., Collino, A., Ghisletti, S., et al. (2013). Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell 153, 101–111.

Halic, M., Gartmann, M., Schlenker, O., Mielke, T., Pool, M.R., Sinning, I., and Beckmann, R. (2006). Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312, 745–747. Hancks, D.C., and Kazazian, H.H., Jr. (2012). Active human retrotransposons: variation and disease. Curr. Opin. Genet. Dev. 22, 191–203. Heras, S.R., Macias, S., Ca´ceres, J.F., and Garcia-Perez, J.L. (2014). Control of mammalian retrotransposons by cellular RNA processing activities. Mob. Genet. Elements 4, e28439. Hormozdiari, F., Konkel, M.K., Prado-Martinez, J., Chiatante, G., Herraez, I.H., Walker, J.A., Nelson, B., Alkan, C., Sudmant, P.H., Huddleston, J., et al.; Great Ape Genome Project (2013). Rates and patterns of great ape retrotransposition. Proc. Natl. Acad. Sci. U S A 110, 13457–13462. Jurka, J. (1997). Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons. Proc. Natl. Acad. Sci. U S A 94, 1872– 1877. Kempf, G., Wild, K., and Sinning, I. (2014). Structure of the complete bacterial SRP Alu domain. Nucleic Acids Res. 42, 12284–12294. Khazina, E., Truffault, V., Bu¨ttner, R., Schmidt, S., Coles, M., and Weichenrieder, O. (2011). Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition. Nat. Struct. Mol. Biol. 18, 1006–1014.

12 Molecular Cell 60, 1–13, December 3, 2015 ª2015 Elsevier Inc.

Stewart, C., Kural, D., Stro¨mberg, M.P., Walker, J.A., Konkel, M.K., Stu¨tz, A.M., Urban, A.E., Grubert, F., Lam, H.Y., Lee, W.P., et al.; 1000 Genomes Project (2011). A comprehensive map of mobile element insertion polymorphisms in humans. PLoS Genet. 7, e1002236. Upton, K.R., Gerhardt, D.J., Jesuadian, J.S., Richardson, S.R., Sa´nchezLuque, F.J., Bodea, G.O., Ewing, A.D., Salvador-Palomeque, C., van der Knaap, M.S., Brennan, P.M., et al. (2015). Ubiquitous L1 mosaicism in hippocampal neurons. Cell 161, 228–239. Voorhees, R.M., and Hegde, R.S. (2015). Structures of the scanning and engaged states of the mammalian SRP-ribosome complex. eLife 4, e07975. Voorhees, R.M., and Ramakrishnan, V. (2013). Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236. Walter, P., and Blobel, G. (1982). Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299, 691–698. Wei, W., Gilbert, N., Ooi, S.L., Lawler, J.F., Ostertag, E.M., Kazazian, H.H., Boeke, J.D., and Moran, J.V. (2001). Human L1 retrotransposition: cis preference versus trans complementation. Mol. Cell. Biol. 21, 1429– 1439.

Please cite this article in press as: Ahl et al., Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.003

Weichenrieder, O., Kapp, U., Cusack, S., and Strub, K. (1997). Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14. RNA 3, 1262–1274. Weichenrieder, O., Wild, K., Strub, K., and Cusack, S. (2000). Structure and assembly of the Alu domain of the mammalian signal recognition particle. Nature 408, 167–173. Weichenrieder, O., Stehlin, C., Kapp, U., Birse, D.E., Timmins, P.A., Strub, K., and Cusack, S. (2001). Hierarchical assembly of the Alu domain of the mammalian signal recognition particle. RNA 7, 731–740. Weichenrieder, O., Repanas, K., and Perrakis, A. (2004). Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon. Structure 12, 975–986.

Wild, K., Weichenrieder, O., Strub, K., Sinning, I., and Cusack, S. (2002). Towards the structure of the mammalian signal recognition particle. Curr. Opin. Struct. Biol. 12, 72–81. Wilson, D.N., and Beckmann, R. (2011). The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling. Curr. Opin. Struct. Biol. 21, 274–282. Witherspoon, D.J., Zhang, Y., Xing, J., Watkins, W.S., Ha, H., Batzer, M.A., and Jorde, L.B. (2013). Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations. Genome Res. 23, 1170– 1181.

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