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A Genomewide Search for Ribozymes Reveals an HDV-Like Sequence In

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A Genomewide Search for Ribozymes Reveals an HDV-Like Sequence In REPORTS selection, self-cleavage could be detected in the A Genomewide Search for Ribozymes library. Activity increased up to round 12, when the library became dominated by a small number Reveals an HDV-Like Sequence of active sequences that exhibited robust self- cleavage in the presence of physiological con- 2þ centrations of Mg (0.5 to 1 mM MgCl2). The in the Human CPEB3 Gene concatemeric RNA of this and preceding active rounds cleaved to form fragments that differed Kourosh Salehi-Ashtiani,1,2* Andrej Lupta´k,1* Alexander Litovchick,1 Jack W. Szostak1† in length by about 200 nt, the unit size of the Ribozymes are thought to have played a pivotal role in the early evolution of life, but relatively library. When the kinetically trapped trimers and few have been identified in modern organisms. We performed an in vitro selection aimed at dimers were isolated and renatured, they self- isolating self-cleaving RNAs from the human genome. The selection yielded several ribozymes, one cleaved to yield smaller products (fig. S1). Thus, of which is a conserved mammalian sequence that resides in an intron of the CPEB3 gene, which the selected library displayed properties in belongs to a family of genes regulating messenger RNA polyadenylation. The CPEB3 ribozyme is accordance with the experimental design and structurally and biochemically related to the human hepatitis delta virus (HDV) ribozymes. The consistent with the presence of a single cleavage occurrence of this ribozyme exclusively in mammals suggests that it may have evolved as recently site per monomeric unit. as 200 million years ago. We postulate that HDV arose from the human transcriptome. Cloning and sequencing of the round 12 library (fig. S2) revealed four self-cleaving ribo- NAs play diverse roles in biology, in- transcripts of single genes or RNA genomes of zymes associated with the following human cluding a variety of regulatory and cata- pathogens. Notably, GlmS was first identified as genes: olfactory receptor OR4K15, insulin-like R lytic functions that together provide a riboswitch through computational analysis of growth factor 1 receptor (IGF1R), a LINE 1 support for the BRNA world[ hypothesis (1). conserved bacterial RNA structures and was retroposon, and the cytoplasmic polyadenyla- Catalytic RNAs, or ribozymes, include natural- later observed to self-cleave in the presence of a tion element–binding protein 3 (CPEB3)(Fig. ly occurring phosphoryl transferases and the cofactor (4). CoTC was identified through de- 1A and fig. S3). We studied the CPEB3 ribo- ribosomal peptidyl transferase (2), as well as in tailed studies of cotranscriptional termination of zyme because it occurs within the transcript of vitro selected RNAs (3). Recent discoveries of b-globin mRNA synthesis (5). a single-copy gene and because it is highly regulatory cellular ribozymes, such as the bac- To identify ribozymes encoded in the human conserved (Fig. 1, B and C). terial cofactor–dependent ribozyme GlmS (4) genome, we devised an in vitro selection scheme The isolated CPEB3 clones (including clones and the eukaryotic cotranscriptional cleavage for the isolation of self-cleaving sequences with- from round 10 and 11) originated from 12 (CoTC) ribozyme (5), have raised interesting out imposing fixed cleavage target sites. In this independent progenitor sequences (i.e., indepen- questions about the prevalence and evolution of scheme, a genomic library is constructed in which dent DNase I fragments) of the library (fig. S4). catalytic RNAs in higher eukaryotes. library elements are uniform in size EÈ150 The region of sequence overlap contains the Most of the reactions carried out by naturally nucleotides (nt)^ and are flanked by polymerase minimal sequence necessary for self-cleavage occurring ribozymes involve the chemistry of chain reaction (PCR) primer sequences Efig. S1 activity and also delineates the local conserva- the phosphodiester RNA backbone. Among (11)^. The library was converted into a single- tion boundaries among different species in this phosphotransferases, self-cleaving ribozymes stranded form, circularized by splint-ligation, and region of the intron (Fig. 1, C and E). The se- form perhaps the most diverse subgroup, which converted back to double-stranded DNA to form quence alignment also revealed several muta- includes the hammerhead (6), hairpin (7), and a Brelaxed[ double-stranded circle that can be in tions that are not present in the genome. Rather HDV ribozymes (8); the Neurospora Varkud vitro transcribed (11). Rolling-circle transcription than having randomly accumulated during the satellite (VS) motif (9); the bacterial GlmS produced tandemly repeated RNAs that preserved repeated PCR amplification cycles, some of these ribozyme (4); the eukaryotic CoTC motif (5); the covalent linkage of the entire transcribed mutations appear to have been highly selected and the group I intron–like ribozyme GIR 1 sequence, even after self-cleavage (12). If a se- (Fig. 1E and fig. S5). To rule out the possibility (10). Only two of these are associated with quence encoded a self-cleaving motif, it could that the cleavage activity depends on the muta- mammals: the CoTC motif, the only known self-cleave under appropriate conditions to tions that had been accumulated in the course of self-cleaving human ribozyme, with orthologs produce unit-length copies, as well as kinetically the selection, we tested the activity of RNA in other primates (5), and the HDV ribozyme, trapped misfolded intermediates such as dimers, transcribed from a genomic DNA segment am- the only known self-cleaving ribozyme encoded trimers, and so on. The size difference between plified directly from human genomic DNA. We by a human pathogen, with no known evolu- the cleaved multimers and uncleaved sequences tested the putative ribozyme sequence for self- tionary orthologs. The sparse occurrence of self- provided a positive selection criterion for the cleavage in the absence of the selection PCR cleaving motifs in humans, and mammals in enrichment of active molecules. Dimers, which primer sequences and demonstrated that the general, could indicate that this class of catalytic contain one intact copy of the sequence, were 209-nt genomic sequence alone was sufficient to RNA has been lost over the course of evolution isolated by polyacrylamide gel electrophoresis carry out self-cleavage (Fig. 1G). Additional 5¶ or, alternatively, that these RNAs have escaped (PAGE) purification, reverse-transcribed, and and 3¶ flanking sequences do not interfere with detection. All self-cleaving ribozymes found to amplified to reinitiate the cycle. the activity of the ribozyme, measured as a date were discovered by careful analysis of We started the selection with about 580 ng first-order rate constant (kobs)oftheRNAself- 15 0 T –1 of RNA (or 10 nucleotides) in concatemeric cleavage reaction (kobs 0.69 0.03 hour , form. We allowed the RNA to self-cleave for 1 t 0 1.00 T 0.04 hours). Furthermore, we 1Howard Hughes Medical Institute, Department of Molec- 1/2 ular Biology, and Center for Computational and Integrative hour in the presence of 5 mM MgCl2 at near- designed a set of primers to amplify a 587– Biology (CCIB), 7215 Simches Research Center, Massachu- physiological monovalent salt concentration, base pair (bp) region that included the CPEB3 setts General Hospital, 185 Cambridge Street, Boston, MA pH, and temperature (140 mM KCl, 10 mM ribozyme and È250 bp of flanking sequences 2 02114, USA. Center for Cancer Systems Biology (CCSB) NaCl, 50 mM Tris-HCl, pH 7.4, 37-C). After on each side. Transcripts made from this and Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA. separation by denaturing PAGE, RNA fragments genomic segment showed self-cleavage (Fig. È *These authors contributed equally to this work. corresponding to dimers ( 400 nt) were isolated, 1H). Conversely, shortening the ribozyme to †To whom correspondence should be addressed. E-mail: reverse-transcribed, and amplified to reinitiate the the region of highest conservation allowed us to [email protected] cycle as outlined above. After six rounds of narrow the sequence to 81 nt, to map precisely 1788 22 SEPTEMBER 2006 VOL 313 SCIENCE www.sciencemag.org REPORTS the site of cleavage, and to analyze the products hydroxyl (Fig. 1I). We confirmed the identity nucleophilic attack of a 2¶ hydroxyl on the ad- of self-cleavage. The 81-nt construct cleaved to of the products of self-cleavage by noting that jacent phosphate, which yields a 2¶-3¶ cyclic yield fragments of 9 nt upstream and 72 nt the downstream sequence could be phosphoryl- phosphate and a 5¶ terminal hydroxyl. downstream of the cleavage site. Incubation of ated at its 5¶ end with PNK and that the up- BLAST and BLAT analyses (13, 14)dem- the products of the transesterification reaction stream sequence was not a substrate for T4 onstrate that the CPEB3 ribozyme sequence, with polynucleotide kinase (PNK), in the ab- RNA ligase. Altogether these experiments dem- like the CPEB3 gene itself, is found as a single sence of adenosine triphosphate, shifted the onstrate that the 72-nt core of the sequence is copy in the genome and is highly conserved in mobility of the upstream 9-nt fragment; this sufficient to carry out self-cleavage, that the all mammalian species examined, including change suggested that the upstream fragment ribozyme can function in the presence of its opossum (a marsupial) (Fig. 1F). The ribozyme contains a 2¶-3¶ cyclic phosphate and that the native flanking sequences, and that the chem- resides in a large intron (È46 kbp in human, downstream 72-nt fragment has a 5¶ terminal istry of self-cleavage most likely proceeds via a È37 kbp in rat, and È35 kbp in mouse) about Fig. 1. Mapping and activity of the human CPEB3 ribo- zyme. (A) Primary structure of CPEB3 (also KIAA0940; GenBank NM_014912) pro- tein (Q, glutamine-rich do- main; RRM, RNA-binding domains; Znf, zinc finger; and aa, amino acid) and mRNA.
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