domain into an extended stem-loop structure ...

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Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure. T S Rozhdestvensky, A M Kopylov, J Brosius, et al. RNA 2001 7: 722-730

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© 2001 RNA Society

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RNA (2001), 7 :722–730+ Cambridge University Press+ Printed in the USA+ Copyright © 2001 RNA Society+

Neuronal BC1 RNA structure: Evolutionary conversion of a tRNAAla domain into an extended stem-loop structure

TIMOFEY S. ROZHDESTVENSKY,1,2 ALEXEI M. KOPYLOV,2 JÜRGEN BROSIUS,1 and ALEXANDER HÜTTENHOFER 1 1 2

Institute of Experimental Pathology/Molecular Neurobiology, D-48149 Münster, Germany Chemistry Department, Moscow State University, 119899, Moscow, Russian Federation

ABSTRACT By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brainspecific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 59 domain of BC1 RNA, derived from tRNAAla , does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNAAla domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 39 end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA. Keywords: BC1 RNA; chemical probing; ID elements; neuronal dendritic RNA; secondary structure; tRNA ancestor

short interspersed repetitive elements (SINEs)+ ID sequences are, similar to Alu sequences in primates, copies of short transcripts that, after reverse transcription into cDNA, are randomly integrated into genomes+ Consequently, large numbers of SINEs accumulate in genomes over time; most of them will have no selective advantage and decay in the genomic environment, although a few can be considered migratory regulatory elements as they may alter expression of targeted genes in a number of ways (Brosius & Gould, 1992; Brosius & Tiedge, 1995; Britten, 1997; Brosius, 1999b, 1999c; Makalowski, 2000)+ There are an estimated one million Alu elements in the human genome and ID elements populate rodent genomes at up to 130,000 copies per haploid genome (Deininger et al+, 1996)+ Within a species, ID sequences exhibit a large degree of sequence similarity to their cognate BC1 RNA (59 domain)+ In contrast, there are greater interspecies differences between BC1 RNA and ID elements+ Based on these observations, we established that BC1 RNA is the first known master gene of a SINE (Kim et al+, 1994)+ In some rodents, such as rat, one or several transcribed ID elements served as additional and highly active master genes for ID elements (Deininger et al+, 1996)+ The evolutionary sequence of

INTRODUCTION A small, brain-specific non-messenger RNA designated BC1 RNA arose 60–110 million years ago by retroposition of tRNAAla in rodents (DeChiara & Brosius, 1987)+ Retroposition is an ancient (Brosius, 1999a) but ongoing process (Brosius, 1999b) that reverse transcribes cellular RNA into cDNA followed by random integration into the genome+ Despite its relatively young age, the BC1 RNA coding region was found to be conserved at significantly higher levels compared to flanking regions when loci of various rodents were analyzed (Martignetti & Brosius, 1993a)+ Therefore, the RNA must have been recruited or exapted (Brosius & Gould, 1992) into a—with respect to the founder tRNA—variant or novel function, as it cannot function as bona fide tRNA+ This is, among other things, a consequence of a missing CCA end+ The 152-nt coding region of rat BC1 RNA has a tripartite structure+ The tRNA-derived domain at the 59 end is not only similar to tRNAAla (close to 75%) but also to rodent ID sequences (Sutcliffe et al+, 1982),

Reprint requests to: Jürgen Brosius, Institute of Experimental Pathology/Molecular Neurobiology, Von-Esmarch-Str+ 56, 48149 Münster, Germany; e-mail: RNA+world@uni-muenster+de+

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Neuronal BC1 RNA structure events in rodents is thus: tRNAAla r BC1 RNA r ID elements+ In BC1 RNA the 59 ID or tRNA-like domain is followed by an adenosine-rich region of about 50 nt and a so-called unique region, as it is nonrepetitive, of about 25 nt in length+ Although the vast majority of SINEs including ID elements are transcriptionally incompetent in vivo, BC1 RNA is expressed from a single active gene on the distal arm of mouse chromosome 7 (Taylor et al+, 1997)+ Unlike most other RNA polymerase III transcripts, expression is almost exclusively restricted to nerve cells in an activity-dependent manner (Muslimov et al+, 1998) and at significantly lower levels in the germ line, such as immature spermatogonia in testes (H+ Tiedge, pers+ comm+)+ Again, unlike most cellular RNAs, BC1 RNA is transported into dendritic processes (Tiedge et al+, 1991) where it may, complexed with proteins as an RNP (Kobayashi et al+, 1991; Cheng et al+, 1996), exert its function (Brosius & Tiedge, 1995, 2001)+ Few other RNA species, including selected mRNAs, tRNAs, and other components of the translational apparatus, can be found in distal segments of dendrites (Tiedge & Brosius, 1996)+ It is thought that local and regulated translation of selected mRNAs near synapses is one of the molecular mechanisms underlying learning and memory (Huang, 1999; Tiedge et al+, 1999)+ Mice with a targeted deletion of the BC1 gene appeared to exhibit behavioral differences when compared to wild-type mice (B+V+ Skryabin, H+ Prior, A+ Güntürkün, L+ Lewejohann, N+ Sachser, A+L+ Vyssotski, H+-P+ Lipp, U+ Jordan, I+A+ Muslimov, H+ Tiedge, & J+ Brosius, in prep+)+ As the 59 domain of BC1 RNA is derived from a tRNA, the RNP may be involved in modulation of cellular activities similar to the ones of its ancestor and therefore still may play a role related to translation+ Alternatively, the 59 domain could serve as a determinant for dendritic transport, as tRNA itself can be transported into dendrites (Tiedge & Brosius, 1996) whereas the central and/or 39 domains may represent the functional domains+ Secondary structure information about BC1 RNA is a basic requirement for obtaining some answers to these questions+ Consequently, we investigated the secondary structure of BC1 RNA by chemical and enzymatic probing+ For comparison, we made an analogous construct, where the 59 domain of BC1 RNA has been exchanged with one that is based on the sequence of an ancestral tRNAAla domain+ RESULTS AND DISCUSSION We performed chemical probing on the 59 region of BC1 RNA (Fig+ 1A,C) to examine whether the tRNAAla element of BC1 RNA would fold into a tRNA-like domain or into an alternate structure+ We compared the data to those obtained with an RNA construct in which the tRNAAla -like domain of BC1 was replaced with an authentic rodent tRNAAla , designated as tala-BC1 RNA

723 (Fig+ 1B)+ The study was carried out by applying specific chemical probes that modify bases not involved in a Watson–Crick interaction: 1-cyclohexyl-3-(2morpholinoethyl)-carbodiimide metho-p -toluene-sulfonate (CMCT) modifying uracil (at N3), dimethyl sulfate (DMS) modifying adenine (at N1), cytosine (at N3), and guanine (at N7), as well as kethoxal (KE) modifying guanine (at N1 and N2)+ The N7 position of guanine bases, modified by DMS, is not involved in a Watson– Crick interaction+ Modified bases within both RNAs were detected by primer extension analysis (see Materials and Methods)+ Probing data of BC1 RNA (152 nt in size) were superimposed onto the BC1 RNA 59 domain that was forced to fold as a canonical tRNA and compared to that of the predicted tala-BC1 construct (155 nt in size)+ The experimentally obtained probing data of the 59 domain of BC1 RNA are inconsistent with the canonical cloverleaf structure of tRNAs (Fig+ 1)+ When compared to tala-BC1 RNA, the most obvious differences in base reactivities in BC1 RNA are found within regions that would correspond to the anticodon loop and T-loop (Fig+ 1)+ Within the structure corresponding to the anticodon loop, all bases of tala-BC1 RNA are modified by base-specific probes+ Therefore, these bases are not involved in Watson–Crick interactions, a finding that is consistent with the predicted anticodon loop structure (Fig+ 1B,D)+ In contrast, within the corresponding sequence of the BC1 RNA domain, only bases C37U38A39 are reactive whereas the remaining bases in the predicted loop are not (Fig+ 1A,C)+ This supports that the sequence in question participates in an alternate structure in BC1 RNA+ Within the loop structure of the pseudouridine arm of tala-BC1 RNA, bases U60 , G62 , and A63 are accessible to chemical probes (Fig+ 1B,D), whereas C61 is reactive in BC1 RNA (Fig+ 1A,C)+ Within the D arm of BC1 RNA, A28 would be predicted to base pair with U18 according to the canonical tRNA cloverleaf structure (Fig+ 1A), which is inconsistent with the observed strong reactivity of A28 to DMS within BC1 RNA (Fig+ 1A,C)+ Importantly, the predicted acceptor stem of the BC1 tRNA domain would be unable to form due to six base substitutions within this region as compared to authentic tRNAAla (Fig+ 1A)+ Accordingly, bases within the acceptor stem should be reactive towards chemical probes KE, DMS, and CMCT+ However, we cannot observe any modifications of bases within this region of BC1 RNA, observations that again are consistent with an alternate structure (see discussion below)+ We noted that analysis of modified nucleotides by primer extension in a region of the BC1 RNA 59 domain extending from position A26 to G35 (Fig+ 1A,C) was hampered by a gel compression of DNA fragments+ This resulted in difficulties interpreting the probing data, particularly at position A28 , which appeared to be a strongly modified base+ To resolve this ambi-

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FIGURE 1. Secondary structure analysis of the 59 ID domain of BC1 RNA (A and C) as compared to tala-BC1 RNA (B and D) by chemical probing+ A: Predicted structure of the 59 domain of BC1 RNA if the canonical cloverleaf structure of tRNAAla is to be maintained+ Base changes in BC1 RNA compared to the authentic tRNAAla are shown by red lettering+ B: Secondary structure of tala-BC1 RNA, in which the 59 ID domain of BC1 RNA is replaced by a canonical tRNAAla + Strong, medium, and weak reactivities of bases towards chemical probes KE, DMS, and CMCT are indicated by red, blue, and green circles, respectively+ C: Chemical probing of the 59 region (ID-region) of BC1 RNA with KE, DMS, or CMCT+ U, G, C, and A: sequencing lanes; contr+: control lane, no chemical probes added+ D: Chemical probing analysis of tala-BC1 RNA with KE, DMS, or CMCT+ U, G, C, and A: sequencing lanes; contr+: control lane, no chemical probes added+

guity, we constructed two BC1 mutants to alleviate the compression+ As observed previously by sequencing BC1 cDNA clones (DeChiara & Brosius, 1987), the compression is caused by the G29C30G31C32 se-

quence (Fig+ 2D)+ In the first mutant, C30 was replaced by G30 and in the second mutant, C32 was replaced by G32 + As a result of both substitutions, we were able to resolve the region prone to compression

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Neuronal BC1 RNA structure

FIGURE 2. Chemical and enzymatic probing of BC1 RNA (A, B, and C) and predicted secondary structure model of BC1 RNA (D)+ A: Probing of BC1 RNA with KE, DMS, and CMCT and V1 nuclease; U, G, C, and A: sequencing lanes; contr+: control lane, no probes added+ B: Chemical probing of the 39 region (unique region) of BC1 RNA with KE, DMS, and CMCT+ U, G, C, and A: sequencing lanes; contr+: control lane, no chemical probes added; reactivities of bases towards chemical probes are indicated by circles+ C: Probing of BC1 RNA with lead acetate+ U, G, C, and A: sequencing lanes; contr+: control lane, no lead acetate added+ 1: plus 5 mM Pb 21 ; 2: plus 7+5 mM Pb 21 ; 3: plus 10 mM Pb 21 ; 4: plus 15 mM Pb 21 + D: Secondary structure model of BC1 RNA and summary of chemical and enzymatic probing data+ Strong, medium, and weak reactivities of bases towards chemical probes (KE, DMS, CMCT) are indicated by red, blue, and green circles, respectively+ Lead acetate cleavage is shown by black arrows, cleavage by V1 cobra venom ribonuclease is indicated by green arrows+ Medium reactivities of bases towards chemical probes (KE, DMS, CMCT) within the poly-A-rich region are indicated by a blue frame+

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726 and unambiguously demonstrate strong modification of A28 within that region (data not shown)+ Based on our data, we propose a secondary structure model for the 59 domain of BC1 RNA different from tRNA+ We suggest that during the course of evolution, the cloverleaf structure of canonical tRNAAla within BC1 RNA underwent a transition into an extended stemloop structure exhibiting three internal bulges (Fig+ 2D)+ In fact, within the proposed bulges, strong modification of bases U22 , A28 , and A49 , and weaker modifications of bases A48 , G50 , and C61 were experimentally demonstrated+ Also, bases within the proposed loop region of BC1 RNA (C37U38A39 ) were accessible to chemical probes, again consistent with the proposed secondary structure model+ To substantiate our data, we performed lead acetate cleavage of BC1 RNA as well as enzymatic probing by V1 nuclease+ Probing of BC1 RNA with lead acetate shows cleavage sites between positions U12 to A15 , G21 to G23 , G35 to C37 , U38 to C41 , A48 to G51 , and U59 to U64 (Fig+ 2C,D)+ Lead cleavage usually occurs at bulges or flexible regions within RNAs (Hüttenhofer et al+, 1996)+ The resulting data correlate well with our secondary structure model+ Nuclease V1 preferentially cleaves RNA at double-stranded or highly structured regions (Fig+ 2A,D; Mougel et al+, 1987)+ In fact, the proposed extended stem structure within the 59 domain of BC1 as well as the three internal bulges are confirmed by V1 nuclease probing (Fig+ 2A,D)+ In addition to chemical and enzymatic probing studies, the proposed secondary structure model is supported by computeraided folding of BC1 RNA, confirming the extended stem-loop structure suggested by our probing experiments (mfold version 3+0 by Zuker and Turner, Depart-

FIGURE 3. Base substitutions in the 59 domain of BC1 RNA within different rodent species (guinea pig, squirrel, chinese hamster) compared to rat BC1 RNA+ Substitutions are indicated by arrows as well as circles, squares or triangles, respectively+ At position 74, a base substitution from C to U is found in chinese hamster, syrian hamster, mouse, californian mouse, deer mouse, squirrel, and gerbil+

T.S. Rozhdestvensky et al. ment of Mathematical Sciences, 331 Amos Eaton Hall, Rensselaer Polytechnic Institute, Troy, New York 121803590, USA; see Materials and Methods)+ In the predicted secondary structure of the 59 stem we did not yet detect any confirming compensatory changes in other rodent sequences of BC1 RNA+ However, the extended stem structure is not, by and large, contradicted by phylogenetic data (Fig+ 3): There are 14 positions that are altered in guinea pig, squirrel, hamster, mouse, californian mouse, deer mouse, and gerbil (Martignetti & Brosius, 1993a; Kass et al+, 1996)+ Three changes are located in single-stranded areas+ Of the 11 substitutions in predicted double-stranded areas, 9 maintain existing base pairs, however, without a compensatory mutation on the other strand, such as from G-U to G-C, G-U to A-U, or vice versa+ One double substitution in guinea pig changes the G29-C47 pair to A-G+ Only two base changes (in squirrel A to C, corresponding to position 43 in rat, as well as in guinea pig G to U, corresponding to position 46 in rat) lead to a potentially destabilizing C-U juxtaposition (Fig+ 3)+ These changes may affect some details of the structure, but not the structure as a whole+ Which nucleotide changes were necessary for the transition of the cloverleaf structure of tRNAAla into the rod-like structure of the BC1 RNA 59 domain? One hypothetical scenario is that BC1 RNA was the retropositional product of a precursor tRNAAla rather than a mature tRNAAla , accounting for the 59 extension of BC1 RNA (Fig+ 4)+ This 6-nt expansion may have been an important precondition to forming the extended structure of BC1 RNA+ One of two changes in this extension (C2 to G2 ) was presumably important to stabilize the BC1 RNA structure+ Equally important was destabiliza-

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Neuronal BC1 RNA structure

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FIGURE 4. Model for tRNAAla cloverleaf ancestral structure conversion into an extended stem-loop structure within BC1 RNA+ Base changes between BC1 RNA and its putative tRNAAla precursor are indicated by circles+ To facilitate comparison, the bases corresponding to the acceptor-, D-, T-, and anticodon arms of tRNAAla are highlighted in red, blue, green, or orange, respectively+

tion of the tRNA acceptor stem through five nucleotide substitutions (positions 72, 73, 75, 76, 77; numbering as in Fig+ 4)+ At the same time, the two proximal substitutions (positions 72, 73) stabilized the extended BC1 structure+ Of the remaining 15 nucleotide substitutions in the mature tRNAAla , only few were likely to be important for further destabilizing the cloverleaf structure+ On the other hand, six substitutions (positions 13, 44, 46, 47, 55, and 69) provided additional stability to the rod-like BC1 RNA structure+ This scenario is open to testing by attempting to switch structures artificially (from cloverleaf to rod-like) by a minimal amount of base substitutions, thus retracing a path evolution might have taken+ Despite the loss of the tRNA cloverleaf structure in BC1 RNA, there may be remaining structural elements that could be recognized by proteins that also interact with tRNA+ Interestingly, the most characteristic element of tRNAAla , the G3-U69 identity base pair in the acceptor stem of the tRNA (Gabriel et al+, 1996), although absent in the forced cloverleaf structure of BC1 RNA, is present and correctly placed in the extended stem-loop structure of BC1 RNA, although in guinea pig this pair is replaced by G3-C69 (Figs+ 1A and 4)+ In addition, Sakamoto and Okada (1985) have reported that the cytosine at position 54 of an in vitro-transcribed BC1 RNA related transcript (an ID repetitive element; see the Introduction) could be modified into its 5-methyl derivative using a cell extract+ Substantiation of this

result awaits analysis of BC1 RNA isolated from mouse or rat brain for modified nucleotides+ To examine the entire secondary structure of BC1 RNA, we performed chemical probing of two other domains of BC1 RNA, namely the poly(A)-rich region and the 39 unique region (Fig+ 2D)+ Probing with CMCT, KE, and DMS revealed that all bases of the poly(A)-rich region are reactive towards chemical modification, indicating that bases are, as expected, not involved in Watson–Crick interactions (data not shown)+ Chemical probing of the 39 unique region is consistent with a small stem-loop structure at the 39 end of BC1 RNA (Fig+ 2B,D)+ We have detected strong modifications of bases at positions A133 , A134 , and C135 and weaker modifications at positions G138 and A140 + These results are consistent with an internal bulge and a loop consisting of three bases at the top of the stem (Fig+ 2B,D)+ Bases C145 and A146 , implied to be part of the internal bulge, are not modified, indicating that they might be involved in some other interactions within BC1 RNA+ Therefore, BC1 RNA contains two stem-loop structures forming the 59 and 39 domains, linked via a singlestranded poly(A)-rich region+ Although the central A-rich region and the 39 domain of BC1 RNA are generally more diverged among rodents than the 59 domain, a phylogenetic analysis revealed two conserved primary structure cores at the 39 end (corresponding to positions 124–135 and 145–151 in the rat)+ Parts of these cores form the stem and

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728 bulge region in the 39 domain of BC1 RNA suggested by our chemical probing data (Fig+ 2)+ This structure might form a binding site for protein(s) that interact with BC1 RNA+ CONCLUSIONS BC1 RNA is a small non-messenger RNA of 152 nt in length, exclusively expressed in brain and at significantly lower levels in the germ line of rodents (H+ Tiedge, pers+ comm+)+ Its evolutionary conservation in all rodent species points to a functional role of BC1 RNA within these tissues+ In brain, BC1 RNA is specifically transported into dendritic processes of neurons and discussed to play a role in regulation of translation at these sites (Brosius & Tiedge, 1995)+ The 59 domain of BC1 RNA is derived from tRNAAla (75% sequence similarity) and therefore we addressed the question of whether this part of BC1 RNA could fold into the canonical tRNA cloverleaf structure or into an alternate structure+ By chemical and enzymatic probing, we present evidence that during the course of evolution the 59 domain of BC1 underwent a structural transition into an extended rod-like stem-loop structure that is substantially different from the original tRNA cloverleaf structure (Fig+ 4)+ Nucleotide changes responsible for this structural transition are predominantly located in the sequences corresponding to the acceptor and anticodon stems of authentic tRNAAla , in BC1 RNA participating now in formation of the 59 stem structure+ What is the functional change that accompanied the structural rearrangement of BC1 RNA? At this point, we can only speculate about the function of the tRNAderived domain within BC1 RNA+ Because BC1 RNA could not be detected in other mammalian orders, except for a potential analog in anthropoid primates, namely BC200 RNA (Martignetti & Brosius, 1993b; Tiedge et al+, 1993; Skryabin et al+, 1998), it is unlikely that BC1 RNA function is essential for all nerve cells+ It is more likely to modulate an existing function that may be common to all mammals or vertebrates+ Perhaps BC1 RNA and BC200 RNA just contribute to a higher level of efficiency or regulation of biological processes in dendrites of neurons+ Muslimov et al+ (1997) have shown that the extended stem-loop structure is necessary and sufficient for dendritic transport of BC1 RNA+ As genuine tRNAs can be transported into dendritic processes of nerve cells (Tiedge & Brosius, 1996), the structural changes leading to BC1 RNA were probably not necessary for its dendritic localization+ One explanation may be that BC1 is more efficiently transported into dendrites, a feature that would not be desirable for tRNAs, as the bulk of these molecules are required for protein biosynthesis in soma+ The three-dimensional structure of this domain with its cognate binding proteins will be a further prerequisite to answer this question+

MATERIALS AND METHODS Materials All reagents were purchased from Boehringer (Germany); DMS and CMCT were obtained from Fluka AG (Switzerland); KE was purchased from Upjohn (England)+

Construction of DNA fragments used in the synthesis of RNAs The DNA fragment encoding wild-type BC1 RNA was PCR amplified (Mullis & Faloona, 1987) from plasmid pBCX607 (Cheng et al+, 1996), using oligonucleotides BC1-59-ID 1 T7 and Unique Rev+ The DNA fragment encoding BC1 RNA with a 39 tag for primer extension analysis of the BC1 RNA 39 domain was amplified by PCR from plasmid pBC1-tag (S+ Subramaniam, B+ Skryabin, unpubl+), using oligonucleotides BC1-59-ID 1 T7 and BC200-BC1-Tag+ DNA fragments harboring mutations (C30 to G30 or C32 to G32 ) within BC1 RNA were generated by PCR from plasmids pBCX607 using oligonucleotides BC1 mut (C30 to G30 ) and Unique Rev+ or BC1 mut (C32 to G32 ) and Unique Rev+, respectively+ DNA templates for T7 transcription of mutant RNAs were prepared by a second PCR amplification reaction using the above DNA fragments as templates and the oligonucleotides BC1-59-ID 1 T7 and Unique Rev+ The tala-BC1 RNA gene chimera was constructed as follows: a gene for tRNAAla was PCR amplified from mouse genomic DNA by using oligonucleotides tRNAAla -Rev+ and tRNAAla -Forw+ and cloned into the pCR2+1 vector of the TA cloning kit (Invitrogen)+ The poly(A)-rich region and the 39 unique domain of BC1 RNA were generated by PCR using oligonucleotides Adap-Forw+ and Unique Rev+ The AdapForw+ primer contains, in addition to sequences of the poly A-rich region, the 39 portion of tRNAAla + In the next step, tRNAAla was PCR amplified by using tRNAAla -Forw+ and Adap+ Rev+ Both PCR products (tRNAAla and poly A/unique region) were annealed and amplified using tRNAAla -Forw+ and Unique Rev+ oligonucleotides+ The final PCR fragment was cloned into vector pCR2+1, designated as plasmid pBtR-8-1+ For T7 transcription, a DNA fragment was amplified from plasmid pBtR-8-1 using oligonucleotides T71tRNAAla Forw+ and Unique Rev+

Oligonucleotides used for generation of DNA fragments and primer extension analysis All oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany)+ BC1-59-ID 1 T7: 59-TAATACGACTCACTATAGGGGGTTGG GGATTTAGCTCAG TGGTAG-39; Unique Rev+: 59-AAAGGTTGTGTGTGCCAGTTAC-39; BC200-BC1-Tag: 59-AAAUGUUGCUAUAUCUCUCGAA-39; BC1 mut (C32 to G32 ): 59-GGGGATTTAGCTCAGTGGTAGA GCGGTTGCCTAG-39; BC1 mut (C30 to G30 ): 59-GGGGATTTAGCTCAGTGGTAGA GGGCTTGCCTAG-39; tRNAAla -Rev+: 59-TTGGAGATGCCGGGGATCGAA-39;

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Neuronal BC1 RNA structure tRNAAla -Forw+: 59-GCGGCTGGGGATGTAGCTCAG-39; Adap-Rev+: 59-CTTTTTTTTTTTTTTTTTTTTTTCGGAGAT GCCGGGGATCGAAC-39; Adap-Forw+: 59-GTTCGATCCCCGGCATCTCCGAAAAAAA AAAAAAAAAAAAAAAG-39; T71tRNAAla Forw+: 59-TAATACGACTCACTATAGGGCGGCT GGGGATGTAGCTCAG-39; BC1-b: 59-GTCTTTTTTTTTTTTTTTTTTTTTTCGG-39; BC200-BC1-Tag: 59-AAAUGUUGCUAUAUCUCUCGAA-39+

T7 transcription of RNAs RNAs for probing analysis were in vitro-transcribed from DNA templates containing a T7 promoter+ Transcription, using T7 RNA polymerase, was performed directly from PCR-amplified DNA fragments (15 mg) in a reaction volume of 400 mL (Milligan et al+, 1987)+ RNAs were purified on denaturing 8% (w/v) polyacrylamide, 7 M urea gels and passively eluted from the gels in 0+3 M Na-acetate buffer, pH 5+2, containing 1 mM EDTA, 0+2% phenol+ Subsequently, RNAs were EtOH precipitated and dissolved in 20 mL H2O+

Chemical probing Chemical probing (Stern et al+, 1988) of wild-type BC1 RNA or BC1 RNA mutants was performed by the addition of 1+5 mL DMS (1:10 dilution in 95% EtOH), 5 mL KE (1:5 dilution of 37 mg/mL stock solution in H 2O) or CMCT (84 mg/mL in H2O) to the RNA (10–15 pmol) in a 50-mL reaction volume and incubation at 30 8C for 20 min+ Prior to probing, RNAs were denatured by incubation of the RNA at 95 8C for 1 min and renatured by cooling at 4 8C for 5 min+ Reactions were performed in 50 mM sodium cacodylate, pH 7+2 (for probing with KE or DMS) or 50 mM sodium borate, pH 8+3 (for probing with CMCT), and 100 mM KCl, 7 mM MgCl2 + All reactions were stopped by the addition of 300 mL EtOH followed by rapid mixing+ For DMS reactions, 25-mL DMS stop solution (1 M Tris-HCl, pH 7+5, 1 M 2-mercaptoethanol, 0+1 M EDTA) were added prior to addition of EtOH+ KE-modified samples were adjusted to 25 mM sodium borate, pH 7+2+ The pellets were resuspended in 200 mL of 0+3 M sodium acetate, pH 5+2, and extracted once with phenol and chloroform+ After precipitation, pellets were dissolved in 10 mL H2O+ All probing experiments for BC1 RNA were repeated at least five times with high reproducibility+

Lead acetate cleavage Cleavage of RNAs (10–15 pmol) with Pb 21 was performed as described previously (Krzyzosiak et al+, 1988)+ Reactions were done in a 20 mL reaction volume at 30 8C for 5 min by addition of lead acetate in concentrations as specified+ Reactions were stopped by the addition of 5 mL 0+5 M EDTA, pH 8+2, and 50 mL 0+3 M sodium acetate, pH 5+2, followed by two phenol and one chloroform extractions; subsequently, samples were EtOH precipitated and resuspended in 10 mL H2O+

Enzymatic probing with V1 cobra venom ribonuclease Digestion of 10 pmol BC1 RNA with nuclease V1 (concentrations specified in Fig+ 2) was performed in 20 mM Tris-HCl,

729 pH 7+5, 7 mM MgCl2 , and 200 mM KCl in a total volume of 20 mL for 15 min on ice+ Reactions were terminated by addition of 80 mL sodium acetate, pH 5+2, and extracted once with phenol and chloroform, respectively+ After precipitation, pellets were resuspended in 10 mL H 2O+

Primer extension reactions of RNAs modified by chemical or enzymatic probing Primer extension reactions were performed according to Stern et al+ (1988) using 59 32 P-end-labeled primers BC1-b and BC200-BC1-Tag+ Samples were loaded onto 8% (w/v) polyacrylamide, 7 M urea gels+ Electrophoresis was performed at 1600 V, 25 mA for 2 h+

Folding prediction of BC1 RNA structure The secondary structure of BC1 RNA was predicted with the mfold program, version 3+0 by Zuker and Turner (http:// bioinfo+math+rpi+edu/;mfold/rna/form1+cgi)+

ACKNOWLEDGMENTS This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Br754/2) and an Interdisciplinary Centre for Clinical Research (IZKF) grant (Teilprojekt D11, Münster) to J+B+ We thank two anonymous referees for their careful analysis of data and constructive suggestions+

Received December 11, 2000; returned for revision January 16, 2001; revised manuscript received January 31, 2001

REFERENCES Britten RJ+ 1997+ Mobile elements inserted in the distant past have taken on important functions+ Gene 205 :177–182+ Brosius J+ 1999a+ Transmutation of tRNA over time+ Nature Genet 22 :8–9+ Brosius J+ 1999b+ Genomes were forged by massive bombardments with retroelements and retrosequences+ Genetica 107 :209–238+ Brosius J+ 1999c+ RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements+ Gene 238 :115–134+ Brosius J, Gould SJ+ 1992+ On “genomenclature”: A comprehensive (and respectful) taxonomy for pseudogenes and other “junk DNA+” Proc Natl Acad Sci USA 89 :10706–10710+ Brosius J, Tiedge H+ 1995+ Reverse transcriptase: Mediator of genomic plasticity+ Virus Genes 11:163–179+ Brosius J, Tiedge H+ 2001+ Dendritic BC1 RNA: Intracellular transport and activity-dependent modulation+ In: Richter D, ed+ Cell polarity and subcellular RNA localization+ Berlin: Springer Verlag+ pp 129– 138+ Cheng JG, Tiedge H, Brosius J+ 1996+ Identification and characterization of BC1 RNP particles+ DNA Cell Biol 15 :549–559+ DeChiara TM, Brosius J+ 1987+ Neural BC1 RNA: cDNA clones reveal nonrepetitive sequence content+ Proc Natl Acad Sci USA 84 :2624–2628+ Deininger PL, Tiedge H, Kim J, Brosius J+ 1996+ Evolution, expression, and possible function of a master gene for amplification of an interspersed repeated DNA family in rodents+ Prog Nucleic Acid Res Mol Biol 52 :67–88+ Gabriel K, Schneider J, McClain WH+ 1996+ Functional evidence for indirect recognition of G+U in tRNA(Ala) by alanyl-tRNA synthetase+ Science 271:195–197+ Huang EP+ 1999+ Synaptic plasticity: Regulated translation in dendrites+ Curr Biol 9 :168–170+

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730 Hüttenhofer A, Westhof E, Böck A+ 1996+ Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB+ RNA 2 :354–366+ Kass DH, Kim J, Deininger PL+ 1996+ Sporadic amplification of ID elements in rodents+ J Mol Evol 42 :7–14+ Kim J, Martignetti JA, Shen MR, Brosius J, Deininger P+ 1994+ Rodent BC1 RNA gene as a master gene for ID element amplification+ Proc Natl Acad Sci USA 91:3607–3611+ Kobayashi S, Goto S, Anzai K+ 1991+ Brain-specific small RNA transcript of the identifier sequences is present as a 10S ribonucleoprotein particle+ J Biol Chem 266 :4726– 4730+ Krzyzosiak WJ, Marciniec T, Wiewiorowski M, Romby P, Ebel JP, Giege R+ 1988+ Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe + Biochemistry 27 :5771–5777+ Makalowski W+ 2000+ Genomic scrap yard: How genomes utilize all that junk+ Gene 259 :61– 67+ Martignetti JA, Brosius J+ 1993a+ Neural BC1 RNA as an evolutionary marker: Guinea pig remains a rodent+ Proc Natl Acad Sci USA 90 :9698–9702+ Martignetti JA, Brosius J+ 1993b+ BC200 RNA: A neural RNA polymerase III product encoded by a monomeric Alu element+ Proc Natl Acad Sci USA 90 :11563–11567+ Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC+ 1987+ Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates+ Nucleic Acids Res 15 :8783–8798+ Mougel M, Eyermann F, Westhof E, Romby P, Expert-Bezancon A, Ebel JP, Ehresmann B, Ehresmann C+ 1987+ Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA+ A model for the interaction and the tertiary structure of the RNA binding site+ J Mol Biol 198 :91–107+ Mullis KB, Faloona FA+ 1987+ Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction+ Methods Enzymol 155 :335– 350+

T.S. Rozhdestvensky et al. Muslimov IA, Banker GA, Brosius J, Tiedge H+ 1998+ Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture+ J Cell Biol 141:1601–1611+ Muslimov IA, Santi E, Homel P, Perini S, Higgins D, Tiedge H+ 1997+ RNA transport in dendrites: A cis -acting targeting element is contained within neuronal BC1 RNA+ J Neurosci 17 :4722– 4733+ Sakamoto K, Okada N+ 1985+ 5-Methylcytidylic modification of in vitro transcript from the rat identifier sequence; evidence that the transcript forms a tRNA-like structure+ Nucleic Acids Res 13 :7195– 7206+ Skryabin BV, Kremerskothen J, Vassilacopoulou D, Disotell TR, Kapitonov V, Jurka J, Brosius J+ 1998+ The BC200 RNA gene and its neural expression are conserved in anthropoidea+ J Mol Evol 47 :677– 685+ Stern S, Moazed D, Noller HF+ 1988+ Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension+ Methods Enzymol 164 :481– 489+ Sutcliffe JG, Milner RJ, Bloom FE, Lerner RA+ 1982+ Common 82nucleotide sequence unique to brain RNA+ Proc Natl Acad Sci USA 79 :4942– 4946+ Taylor BA, Navin A, Skryabin BV, Brosius J+ 1997+ Localization of the mouse gene (Bc1 ) encoding neural BC1 RNA near the fibroblast growth factor 3 locus (Fgf3 ) on distal Chromosome 7+ Genomics 144 :153–154+ Tiedge H, Bloom FE, Richter D+ 1999+ RNA, whither goest thou? Science 283 :186–187+ Tiedge H, Brosius J+ 1996+ Translational machinery in dendrites of hippocampal neurons in culture+ J Neurosci 16 :7171–7181+ Tiedge H, Chen W, Brosius J+ 1993+ Primary structure, neuralspecific expression, and dendritic location of human BC200 RNA+ J Neurosci 13 :2382–2390+ Tiedge H, Fremeau RT Jr, Weinstock PH, Arancio O, Brosius J+ 1991+ Dendritic location of neural BC1 RNA+ Proc Natl Acad Sci USA 88 :2093–2097+