Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.     

Non-specific depurination activity of saporin-S6, a ribosome-inactivating protein, under acidic conditions

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.     

Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a mammalian 3′-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), ∼10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.     

In vitro interaction of eukaryotic elongation factor 2 with synthetic oligoribonucleotide that mimics GTPase domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzed the translocation of peptidyl-tRNA from the ribosomal A site to the P site. In this paper, the interaction between eEF2 and GTD RNA, a synthetic oligoribonucleotide that mimicked the GTPase domain of rat 28S ribosomal RNA, was studied in vitro. The purified eEF2 could bind to GTD RNA, forming a stable complex. Transfer RNA competed with GTD RNA in binding to eEF2, whereas poly(A), poly(U) and poly(I, C) did not interfere with the interaction between eEF2 and GTD RNA, demonstrating that the tertiary structure of RNA might be necessary for the recognition of and binding to eEF2. The complex formation of eEF2 with GTD RNA was inhibited by SRD RNA, a synthetic oligoribonucleotide mimic of Sarcin/Ricin domain RNA of rat 28S RNA. Similarly, GTD RNA inhibited the interaction between eEF2 and SRD RNA. This fact implies that these small oligoribonucleotides probably share similar recognition or binding identity elements in their tertiary structures. In addition, the binding of eEF2 to GTD RNA could be obviously weakened by the ADP-ribosylation of eEF2 with diphtheria toxin. These results indicate that eEF2 behaves differently from prokaryotic EF-G in binding to ribosomal RNA.     

The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G

During translocation peptidyl-tRNA moves from the A-site to the P-site and mRNA is displaced by three nucleotides in the 3' direction. This reaction is catalyzed by elongation factor-G (EF-G) and is associated with ribosome-dependent hydrolysis of GTP. The molecular basis of translocation is the most important unsolved problem with respect to ribosome function. A critical question, one that might provide a clue to the mechanism of translocation, is the precise identity of the contacts between EF-G and ribosome components. To make the identification, a covalent bond was formed, by ultraviolet irradiation, between EF-G and a sarcin/ricin domain (SRD) oligoribonucleotide containing 5-iodouridine. The cross-link was established, by mass spectroscopy and by Edman degradation, to be between a tryptophan at position 127 in the G domain in EF-G and either one of two 5-iodouridine nucleotides in the sequence UAG2655U in the SRD. G2655 is a critical identity element for the recognition of the factor's ribosomal binding site. The site of the cross-link provides the first direct evidence that the SRD is in close proximity to the EF-G catalytic center. The proximity suggests that the SRD RNA has a role in the activation of GTP hydrolysis that leads to a transition in the conformation of the factor and to its release from the ribosome.     

Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches

We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.     

Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. Analysis with tetraloop mutants.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.     

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.     

A metalloproteinase associated with gelonin, a "ribosome inactivating protein"

Gelonin, a single-chain protein which inactivates eukaryotic ribosomes, becomes split into peptides when incubated with SDS. During the chromatographic purification of gelonin on carboxymethylcellulose three overlapping peaks emerge in the gelonin elution region, containing three proteins with small differences in apparent molecular weight (31,500, 30,000 and 29,200). All three proteins are endowed with inhibitory activity on protein synthesis and with proteinase activity, although with different specific activities, and all three give rise to the same peptides upon incubation with SDS, suggesting that they are isoforms of gelonin. The gelonin-associated proteinase acts only on gelonin, while it is inactive on the most common substrates for endoproteinases. The proteolytic activity is not inhibited by inhibitors of serine- or SH-proteinases, while it is completely abolished by chelating agents. Divalent cations restore the proteolytic activity inhibited by EDTA. The stability of the proteinase activity on exposure of gelonin to extreme values of pH or to prolonged incubation has been investigated. The inhibitory activity on protein synthesis and the proteinase activity are differently affected by these treatments.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant (Laminaria japonica A).

Lamjapin, a novel type Iota ribosome-inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of approximately 36 kDa, lamjapin is slightly larger than the other known single-chain ribosome-inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC50 of 0.69 nm. It can depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R-fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oligoribonucleotide (35-mer) substrate that mimics the sarcin/ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N-C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome-inactivating proteins from higher plants. We conclude that lamjapin is an RNA N-glycosidase that belongs to the ribosome-inactivating protein family. This study reports for the first time that ribosome-inactivating protein exists in the lower cryptogamic algal plant.     

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues

Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including 11 charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted. Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha-tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase. Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates. Substantial changes in k(cat), K(m)(DMAPP), and/or K(m)(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

To probe functions of the U1 small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas collosoma or Caenorhabditis elegans. In agreement with previous results (J.P. Bruzik and J.A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of U1 snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-O-methyl oligoribonucleotide that disrupts the first stem of U1 snRNA as well as stably sequestering positions of U1 snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-O-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of U1 with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and U1. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the U1 monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of U1 inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of U1 RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of U1 snRNP functions.     

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.     

Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets.

We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'- deoxy oligoribonucleotide probe did not. Due to their enhanced Tm when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets. A 12 base 2'-O-methyl oligoribonucleotide probe had the same Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.     

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.