'Z-DNA like' fragments in RNA: a recurring structural motif with implications for folding, RNA/protein recognition and immune response.

Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme.

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2',3'-cyclic phosphate and 5'-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson-Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme-substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.

Targeting stathmin in prostate cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin is expressed at high levels in a variety of human cancers and provides an attractive molecule to target in cancer therapies that disrupt the mitotic apparatus. We developed replication-deficient bicistronic adenoviral vectors that coexpress green fluorescent protein and ribozymes that target stathmin mRNA. The therapeutic potential of these recombinant adenoviruses was tested in an experimental androgen-independent LNCaP prostate cancer model. Adenovirus-mediated transfer of anti-stathmin ribozymes resulted in efficient transduction and marked inhibition of stathmin expression in these cells. Cells that were transduced with the anti-stathmin adenoviruses showed a dramatic dose-dependent growth inhibition. This was associated with accumulation of LNCaP cells in the G2-M phases of the cell cycle. A similar dose-dependent inhibition of clonogenic potential was also observed in cells infected with anti-stathmin adenoviruses. Morphologic and biochemical analysis of infected cells showed a marked increase in apoptosis characterized by detachment of the cells, increased chromatin condensation, activation of caspase-3, and fragmentation of internucleosomal DNA. If these findings are confirmed in vivo, it may provide an effective approach for the treatment of prostate cancer.

Bacterial Riboswitches and Ribozymes Potently Activate the Human Innate Immune Sensor PKR.

The innate immune system provides the first line of defense against pathogens through the recognition of nonspecific patterns in RNA to protect the cell in a generalized way. The human RNA-activated protein kinase, PKR, is a dsRNA binding protein and an essential sensor in the innate immune response, which recognizes viral and bacterial pathogens through their RNAs. Upon activation via RNA-dependent autophosphorylation, PKR phosphorylates the eukaryotic initiation factor eIF2α, leading to termination of translation. PKR has a well-characterized role in recognizing viral RNA, where it binds long stretches of double-stranded RNA nonsequence specifically to promote activation; however, the mechanism by which bacterial RNA activates PKR and the mode by which self RNA avoids activating PKR are unknown. We characterized activation of PKR by three functional bacterial RNAs with pseudoknots and extensive tertiary structure: the cyclic di-GMP riboswitch, the glmS riboswitch-ribozyme, and the twister ribozyme, two of which are ligand-activated. These RNAs were found to activate PKR with comparable potency to long dsRNA. Enzymatic structure mapping in the absence and presence of PKR reveals a clear PKR footprint and provides a structural basis for how these bacterial RNAs activate PKR. In the case of the cyclic di-GMP riboswitch and the glmS riboswitch-ribozyme, PKR appears to dimerize on the peripheral double-stranded regions of the native RNA tertiary structure. Overall, these results provide new insights into how PKR acts as an innate immune signaling protein for the presence of bacteria and suggest a reason for the apparent absence of protein-free riboswitches and ribozymes in the human genome.

A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination.

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power.

Further characterization of human RNase MRP/RNase P and related autoantibodies.

We characterized a panel of human RNase MRP/RNase P autoantibodies by immunoprecipitation, immunodepletion, immunoaffinity purification and immunoblotting. We report on the protein spectrum that is recognized by RNase MRP/RNase P autoantibodies. We also describe another, related patient serum that based on these assays does not immunoprecipitate RNase P/MRP/Th40. This autoantibody 'KC', however, coimmunoprecipitates the RNase MRP/RNase P associated RNAs from HeLa and La9 cell extracts as shown by nuclease protection experiments.

Characterization of two scleroderma autoimmune antigens that copurify with human ribonuclease P.

Human RNase P has been purified more than 2000-fold from HeLa cells. In addition to the RNA component, H1 RNA, polypeptides of molecular masses 14, 20, 25, 30, 38, and 40 kDa copurify with the enzyme activity. Sera from two different patients with the autoimmune disease scleroderma were used to immunodeplete human RNase P activity. These same sera cross-reacted on immunoblots with two of the copurifying polypeptides, p30 and p38, whereas an autoimmune serum that does not immunodeplete RNase P activity did not react with these proteins. Peptide fragments derived from purified p30 and p38 facilitated the molecular cloning and sequencing of cDNAs coding for these two polypeptides, which are now designated as Rpp30 and Rpp38, respectively. RPP38 cDNA encodes a polypeptide that may be identical to a previously identified antigen of approximately 40 kDa, which is immunoprecipitated by Th and To autoimmune antisera, and that has been implicated as a protein subunit of human RNase P by virtue of its ability to bind to H1 RNA in vitro. The second autoimmune antigen, Rpp30, as such, has not been described previously.

Spinach chloroplast RNase P: a putative protein enzyme.

Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules. In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo. In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA. We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide. Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa. A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA. The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography. Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme. We showed previously that phosphodiester bond cleavage by the E. coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate. In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond. These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme.

A functional site of the GTPase-associated center within 28S ribosomal RNA probed with an anti-RNA autoantibody.

An anti-RNA autoantibody (anti-28S) was employed to identify structural and functional elements characteristic of a domain termed the 'GTPase center' in eukaryotic 28S ribosomal RNA. This antibody, an inhibitor of ribosome-associated GTP hydrolysis, has a unique property: it binds to the RNA domain of eukaryotes but not to that of prokaryotes. The antibody binding occurred in the presence of Mg2+ and protected from chemical modification three conserved bases (U1958, G1960 and A1990) and the base G1959 which is replaced by A in prokaryotic 23S rRNA (A1067 in Escherichia coli). In vitro substitution of G1959 to A drastically weakened the antibody binding, and the reciprocal substitution, A1067-->G of the E.coli domain conferred the binding ability. This suggests that the G base determines the specificity of antibody binding. The G1959 was also protected by the association of ribosomes with elongation factor EF-2. The result, together with protection of E.coli base A1067 by EFG [D.Moazed, I.M. Robertson and H.F. Noller (1988) Nature, 334, 362-364], suggests that the position of G1959 in eukaryotes and A1067 in prokaryotes constitutes at least part of the factor binding site irrespective of the base replacement during evolution.

Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1.

T-cell lines (Jurkat and Molt-4) were transduced with retroviral vectors containing a hairpin ribozyme that targets a conserved sequence in the 5' transcribed leader sequence of human immunodeficiency virus (HIV) type 1. Stable cell lines were generated which constitutively and persistently expressed the ribozyme gene driven by either the Moloney retroviral long terminal repeat (LTR) or an internal human tRNA(val) promoter. There was no apparent deleterious effect of long-term ribozyme expression on cell proliferation or viability. Cells expressing ribozyme were resistant to challenge from diverse strains of HIV, including an uncloned clinical isolate. No reverse transcriptase activity or virus infectivity was detectable in the culture supernatants of Jurkat cells expressing the ribozyme driven by the tRNA(val) promoter up to 35 days after challenge with HIV-1/HXB2. Expression of the ribozyme also significantly decreased (by approximately 50- to 100-fold) the efficiency of incoming virus to synthesize viral DNA. These and previously reported results indicate that transfer and expression of the ribozyme gene interfere with both early and late events in the HIV replication cycle and confer long-term resistance to HIV-1 infection.

hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes.

RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.

Tumor-specific expression of anti-mdr1 ribozyme selectively restores chemosensitivity in multidrug-resistant colon-adenocarcinoma cells.

P-glycoprotein (Pgp)-conferred multidrug resistance (MDR) is expressed in cancer and in normal colon tissues and has important physiological functions. In order to selectively reverse MDR in malignant tissue without disrupting the function of normal colonocytes, a retroviral vector (pCEAMR) containing anti-mdr1 ribozyme coupled to the carcino-embryonic-antigen (CEA) promoter was constructed and introduced into resistant colon-cancer cells (SW1116R) that produce CEA and into control resistant cells (HeLaK) that do not produce CEA. Anti-mdr1 ribozyme was expressed in SW1116R cells but not in HeLaK cells. Subsequently, the expression of mdr1 mRNA and Pgp decreased significantly in the transfected SW1116R cells, and was even lower than in parent non-resistant SW1116 cells. The functional ability of Pgp to facilitate rhodamine 123 (Rh123) efflux showed that the transfected SW1116R cells with low Pgp expression retained Rh123, whereas non-transfected SW1116R cells with high Pgp expression released the dye quickly. There was no difference in mdr1 mRNA or in Pgp between non-transfected and transfected HeLaK cells. Drug resistance to doxorubicin (DOX) decreased 93.1% in the transfected SW1116R cells, while no change in drug resistance occurred in the infected HeLaK cells. DOX could clearly inhibit the growth of transfected SW1116R tumors but had no effect on untransfected and on transfected HeLaK cells in vivo. These results indicate that our anti-mdr1 ribozyme is expressed only in CEA-producing colon-cancer cells and reverses their drug resistance selectively.

Identity of the RNase MRP- and RNase P-associated Th/To autoantigen.

OBJECTIVE: To characterize the molecular identity of the Th/To autoantigen, which is targeted by autoantibodies in scleroderma and which is associated with the human RNase MRP and RNase P ribonucleoprotein complexes. METHODS: Proteins immunoprecipitated by anti-Th/To+ patient antisera from biotinylated total HeLa cell extracts were analyzed by immunoblotting. The association of autoantigenic proteins with the RNase MRP complex was analyzed by reconstitution experiments and ultraviolet crosslinking. The reactivity of patient sera with all known RNase MRP/RNase P proteins was analyzed by immunoprecipitation of the individual recombinant proteins. RESULTS: The previously defined Th40 autoantigen appeared to be identical to the Rpp38 protein. Paradoxically, Rpp38 did not bind to the P3 domain of the RNase MRP RNA, as suggested by previously published data for Th40, and only half of the anti-Th/To+ sera contained anti-Rpp38 reactivity. Two other RNase MRP/RNase P subunits, Rpp20 and Rpp25, were found to interact with the P3 domain. The previously reported 40-kd species associated with this domain appeared to consist of Rpp20 and/or Rpp25 associated with a nuclease-resistant RNA fragment. Finally, we demonstrated that almost all tested anti-Th/To+ patient sera contained autoantibodies to Rpp25 and hPop1, indicating that these proteins harbor the most frequently targeted Th/To determinants. CONCLUSION: Our data unequivocally define the identity of the Th/To autoantigen and demonstrate that Th/To autoepitopes are found on several protein subunits of RNase MRP/RNase P.

Human RNaseP RNA and nucleolar 7-2 RNA share conserved 'To' antigen-binding domains.

RNase P in both prokaryotes and eukaryotes is a ribonucleoprotein that cleaves tRNA precursors to generate the 5' termini of the mature tRNAs. Many patients with autoimmune diseases produce antibodies against a 40 kDa protein (designated To or Th antigen) which is an integral component of eukaryotic RNaseP as well as nucleolar 7-2 RNP which is identical to the mitochondrial RNA processing (MRP) RNP. Interestingly, the To antigen found in human cells and the C5 protein, the only protein component of E. coli RNaseP, are antigenically related. In this study, we show that a 56 nucleotide-long sequence, corresponding to nucleotides 20-75 near the 5' end of human RNaseP RNA, is sufficient to bind the To antigen. We previously showed that the human To antigen binds to a short distinct structural domain near the 5' end of human 7-2/MRP RNA. There is no obvious primary sequence homology between the To antigen binding sites in RNaseP RNA and 7-2/MRP RNA; however, these sequences are capable of assuming a similar secondary structure which corresponds to the recently proposed 'cage' structure for RNaseP RNAs and 7-2/MRP RNA (Forster and Altman (1989) Cell 62: 407-409). These data are supportive of the idea that these two RNAs may have evolved from a common progenitor molecule.

Autoantigenic properties of some protein subunits of catalytically active complexes of human ribonuclease P.

At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.

hPop1: an autoantigenic protein subunit shared by the human RNase P and RNase MRP ribonucleoproteins.

The eukaryotic endonucleases RNase P and RNase MRP require both RNA and protein subunits for function. Even though the human RNase P and MRP RNAs were previously characterized, the protein composition of the particles remains unknown. We have identified a human a Caenorhabditis elegans sequence showing homology to yPop1, a protein subunit of the yeast RNase P and MRP particles. A cDNA containing the complete coding sequence for the human protein, hPop1, was cloned. Sequence analysis identifies three novel sequence motifs, conserved between the human, C. elegans and yeast proteins. Affinity-purified anti-hPop1 antibodies recognize a single 115 kDa protein in HeLa cell nuclear extracts. Immunoprecipitations with different anti-hPop1 antibodies demonstrate an association of hPop1 with the vast majority of the RNase P and MRP RNAs in HeLa cell nuclear extracts. Additionally, anti-hPop1 immunoprecipitates possess RNase P enzymatic activity. These results establish hPop1 as the first identified RNase P and MRP protein subunit from humans. Anti-hPop1 antibodies generate a strong nucleolar and a weaker homogeneous nuclear staining in HeLa cells. A certain class of autoimmune patient serum precipitates in vitro-translated hPop1. hPop1 is therefore an autoantigen in patients suffering from connective tissue diseases.