MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells.

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.

Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells.

Background: Studies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1-2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma. Methods: Synthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C. Results: Ectopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3'UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3'UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence. Conclusions: miR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes.

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P.

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells.

BACKGROUND: Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology. RESULTS: Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation. Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation. CONCLUSIONS: We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.

MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

The MYCN oncogene is frequently amplified in neuroblastoma. It is one of the most consistent markers of bad prognosis for this disease. Dickkopf-3 (DKK3) is a secreted protein of the DKK family of Wnt regulators. It functions as a tumor suppressor in a range of cancers, including neuroblastoma. MYCN was recently found to downregulate DKK3 mRNA. In this study, we show that MYCN knockdown in MYCN-amplified (MNA) neuroblastoma cell lines increases secretion of endogenous DKK3 to the culture media. MicroRNAs (miRNAs) are ∼20 nt long single-stranded RNA molecules that downregulate messenger RNAs by targeting the 3' untranslated region (3'UTR). Many miRNAs regulate genes involved in the pathogenesis of cancer and are extensively deregulated in different tumors. Using miRNA target prediction software, we found several MYCN-regulated miRNAs that could target the 3'UTR sequence of DKK3, including mir-92a, mir-92b and let-7e. Luciferase expression from a reporter vector containing the DKK3-3'UTR was decreased when this construct was cotransfected with mir-92a, mir-92b or let-7e in HEK293 cells. Mutation of the mir-92 seed sequence in the 3'UTR completely rescued the observed decrease in reporter expression when cotransfected with mir-92a and mir-92b. Antagomir and miRNA-mimic transfections in neuroblastoma cell lines confirmed that DKK3 secretion to the culture media is regulated by mir-92. Consistent with reports from other cancers, we found DKK3 to be expressed in the endothelium of primary neuroblastoma samples and to be absent in tumors with MYCN amplification. Our data demonstrate that MYCN-regulated miRNAs are able to modulate the expression of the tumor suppressor DKK3 in neuroblastoma.

Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers. RESULTS: In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced. CONCLUSION: We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA.

DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5'-end of the 3'-cleavage product. The 3'-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5'- and 3'-deletions, we identify a hairpin in the 5'-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5'-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.

Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines.

Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox-doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications.

Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High-risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. The need for biomarkers that can select patients not responding well to therapy in an early phase is therefore needed. In this study, we used next generation sequencing technology to determine the expression profiles in high-risk neuroblastoma cell lines established before and after therapy. Using partial least squares-discriminant analysis (PLS-DA) with least absolute shrinkage and selection operator (LASSO) and leave-one-out cross-validation, we identified a panel of 55 messenger RNAs and 17 long non-coding RNAs (lncRNAs) which were significantly altered in the expression between cell lines isolated from primary and recurrent tumors. From a neuroblastoma patient cohort, we found 20 of the 55 protein-coding genes to be differentially expressed in patients with unfavorable compared with favorable outcome. We further found a twofold increase or decrease in hazard ratios in these genes when comparing patients with unfavorable and favorable outcome. Gene set enrichment analysis (GSEA) revealed that these genes were involved in proliferation, differentiation and regulated by Polycomb group (PcG) proteins. Of the 17 lncRNAs, 3 upregulated (NEAT1, SH3BP5-AS1, NORAD) and 3 downregulated lncRNAs (DUBR, MEG3, DHRS4-AS1) were also found to be differentially expressed in favorable compared with unfavorable outcome. Moreover, using expression profiles on both miRNAs and mRNAs in the same cohort of cell lines, we found 13 downregulated and 18 upregulated experimentally observed miRNA target genes targeted by miR-21, -424 and -30e, -29b, -138, -494, -181a, -34a, -29b, respectively. The advantage of analyzing biomarkers in a clinically relevant neuroblastoma model system enables further studies on the effect of individual genes upon gene perturbation. In summary, this study identified several genes, which may aid in the prediction of response to therapy and tumor recurrence.

Inhibitors of ribosome biogenesis repress the growth of MYCN-amplified neuroblastoma.

Abnormal increases in nucleolar size and number caused by dysregulation of ribosome biogenesis has emerged as a hallmark in the majority of spontaneous cancers. The observed ribosome hyperactivity can be directly induced by the MYC transcription factors controlling the expression of RNA and protein components of the ribosome. Neuroblastoma, a highly malignant childhood tumor of the sympathetic nervous system, is frequently characterized by MYCN gene amplification and high expression of MYCN and c-MYC signature genes. Here, we show a strong correlation between high-risk disease, MYCN expression, poor survival, and ribosome biogenesis in neuroblastoma patients. Treatment of neuroblastoma cells with quarfloxin or CX-5461, two small molecule inhibitors of RNA polymerase I, suppressed MycN expression, induced DNA damage, and activated p53 followed by cell cycle arrest or apoptosis. CX-5461 repressed the growth of established MYCN-amplified neuroblastoma xenograft tumors in nude mice. These findings suggest that inhibition of ribosome biogenesis represent new therapeutic opportunities for children with high-risk neuroblastomas expressing high levels of Myc.

Hsa-miR-376c-3p targets Cyclin D1 and induces G1-cell cycle arrest in neuroblastoma cells.

High-risk neuroblastoma is the most aggressive form of cancer in children. The estimated survival of children with high-risk neuroblastoma is 40-50% compared with low and intermediate risk neuroblastoma, which is >98 and 90-95%, respectively. In addition, patients with high-risk neuroblastoma often experience relapse following intensive treatments with standard chemotherapeutic drugs. Therefore alternative strategies are required to address this problem. MicroRNAs (miRNAs/miRs) are small, endogenously expressed non-coding RNAs, which when deregulated have been demonstrated to serve significant roles in the tumorigenesis of a number of different types of cancer. Results from a previous deep sequencing study identified 22 downregulated miRNAs from the 14q32 miRNA cluster differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse following intensive treatments. miR-376c-3p is one of the 22 miRNAs that was downregulated in the majority of the cell lines isolated from patients post treatment. The present study employed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify the basic expression of miR-376c-3p in 6 neuroblastoma cell lines. The functional role of miR-376c-3p in the neuroblastoma cell lines was evaluated by alamar blue-cell viability and propidium iodide-flow cytometric assays. In addition, luciferase reporter assays, RT-qPCR and western blotting were performed to identify and quantify the targets of miR-376c-3p in neuroblastoma cell lines. Ectopic expression of miR-376c-3p led to significant inhibition of cell viability and G1-cell cycle arrest in multiple neuroblastoma cell lines by reducing the expression of cyclin D1, an oncogene critical for neuroblastoma pathogenesis. The results of the present study provide novel insights into the functional role of miR-376c-3p and suggest new approaches for the treatment of neuroblastoma.

MicroRNA-193b-3p represses neuroblastoma cell growth via downregulation of Cyclin D1, MCL-1 and MYCN.

Neuroblastoma is the most common diagnosed tumor in infants and the second most common extracranial tumor of childhood. The survival rate of patients with high-risk neuroblastoma is still very low despite intensive multimodal treatments. Therefore, new treatment strategies are needed. In recent years, miRNA-based anticancer therapy has received growing attention. Advances in this novel treatment strategy strongly depends on the identification of candidate miRNAs with broad-spectrum antitumor activity. Here, we identify miR-193b as a miRNA with tumor suppressive properties. We show that miR-193b is expressed at low levels in neuroblastoma cell lines and primary tumor samples. Introduction of miR-193b mimics into nine neuroblastoma cell lines with distinct genetic characteristics significantly reduces cell growth in vitro independent of risk factors such as p53 functionality or MYCN amplification. Functionally, miR-193b induces a G1 cell cycle arrest and cell death in neuroblastoma cell lines by reducing the expression of MYCN, Cyclin D1 and MCL-1, three important oncogenes in neuroblastoma of which inhibition has shown promising results in preclinical testing. Therefore, we suggest that miR-193b may represent a new candidate for miRNA-based anticancer therapy in neuroblastoma.

Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron.

BACKGROUND: Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. RESULTS: To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus. CONCLUSION: The Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%), leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.

RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

Next generation sequencing of microRNAs from isogenic neuroblastoma cell lines isolated before and after treatment.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma.

Exosome-like Extracellular Vesicles from MYCN-amplified Neuroblastoma Cells Contain Oncogenic miRNAs.

BACKGROUND: In recent years, evidence has accumulated indicating that both normal and cancer cells communicate via the release and delivery of macromolecules packed into extracellular membrane vesicles. MATERIALS AND METHODS: We isolated nano-sized extracellular vesicles from MYCN-amplified neuroblastoma cell lines using ultracentrifugation and exosome precipitation (Exoquick) protocols. These vesicles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis and western blotting. Exosomal miRNA profiles were obtained using a reverse transcription-polymerase chain reaction (RT-PCR) ready-to-use panel measuring a total of 742 miRNAs. RESULTS: In this study, we showed that MYCN-amplified neuroblastoma cell lines secrete populations of miRNAs inside small exosome-like vesicular particles. These particles were shown to be taken-up by recipient cells. By profiling the miRNA content, we demonstrated high expression of a group of established oncomirs in exosomes from two MYCN-amplified neuroblastoma cell lines. Despite the fact that other studies have demonstrated the ability of exosomal miRNAs both to repress mRNA targets and to stimulate Toll-like receptor-8 (TLR8) signaling in recipient cells, we did not observe these effects with exosomes from MYCN-amplified neuroblastoma cells. However, functional enrichment analysis reveals that mRNA targets of highly expressed exosomal miRNAs are associated with a range of cellular and molecular functions related to cell growth and cell death. CONCLUSION: MYCN-amplified neuroblastoma cell lines secrete exosome-like particles containing oncogenic miRNAs. This work showed for the first time that neuroblastoma cells secrete exosome-like particles containing miRNAs with potential roles in cancer progression. These findings indicate a new way for MYCN-amplified neuroblastoma cells to interact with the tumor environment.

Wnt/ß-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active ß-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment.

DiGIR1 and NaGIR1: naturally occurring group I-like ribozymes with unique core organization and evolved biological role.

The group I-like ribozyme GIR1 is a unique example of a naturally occurring ribozyme with an evolved biological function. GIR1 generates the 5'-end of a nucleolar encoded messenger RNA involved in intron mobility. GIR1 is found as a cis-cleaving ribozyme within two very different rDNA group I introns (twin-ribozyme introns) in distantly related organisms. The Didymium GIR1 (DiGIR1) and Naegleria GIR1 (NaGIR1) share fundamental features in structural organization and reactivity, and display significant differences when compared to the related group I splicing ribozymes. GIR1 lacks the characteristic P1 segment present in all group I splicing ribozymes, it has a novel core organization, and it catalyses two site-specific hydrolytic cleavages rather than splicing. DiGIR1 and NaGIR1 appear to have originated from eubacterial group I introns in order to fulfil a common biological challenge: the expression of a protein encoding gene in a nucleolar context.

Optimization and application of the group I ribozyme trans-splicing reaction.

Group I ribozymes are naturally occurring catalytic RNAs that are able to excise themselves as introns (group I introns) from a precursor RNA, and to ligate the flanking exons. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence specific manner, and to covalently link an RNA sequence to this separate RNA molecule. This ribozyme transesterification reaction has potential in molecular biology and in medicine as a new approach to gene therapy. Here we describe detailed optimized protocols where trans-splicing group I ribozymes are applied in mapping accessible sites in target messenger RNA, and in messenger RNA-repair by correcting mutations.