Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma.

BACKGROUND: MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation. METHODS: A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line. RESULTS: The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells. CONCLUSION: The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme.

DiGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme group I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5'-end of the intron endo-nuclease open reading frame and is a unique example of a group I ribozyme with an evolved biological function. DiGIR1 is the smallest functional group I ribozyme known from nature and has an unusual core organization including the 6 bp P15 pseudoknot. Here we report results of functional and structural analyses that identify RNA elements critical for hydrolysis outside the DiGIR1 ribozyme core moiety. Results from deletion analysis, disruption/compensation mutagenesis and RNA structure probing analysis all support the existence of two new segments, named P2 and P2.1, involved in the hydrolysis of DiGIR1. Significant decreases in the hydrolysis rate, k (obs), were observed in disruption mutants involving both segments. These effects were restored by compensatory base pairing mutants. The possible role of P2 is to tether the ribozyme core, whereas P2.1 appears to be more directly involved in catalysis.

Group I twintrons: genetic elements in myxomycete and schizopyrenid amoeboflagellate ribosomal DNAs.

Protists are unicellular eukaryotes which represent a significant fraction of the global biodiversity. The myxomycete Didymium and the schizopyrenid amoeboflagellate Naegleria are distantly related protists. However, we have noted several striking similarities in life cycle, cell morphology, and ribosomal DNA organization between these organisms. Both have multicopy nuclear extrachromosomal ribosomal DNAs. Here the small subunit ribosomal RNA genes are interrupted by an optional group I twintron, a novel category among the group I introns. Group I twintrons are mobile self-splicing introns of 1.3-1.4 kb in size, with a complex organization at the RNA level. A group I twintron consists of two distinct ribozymes (catalytic RNAs) with different functions in RNA processing, and an open reading frame encoding a functional homing endonuclease--all with prospects of application as molecular tools in biotechnology. Updated RNA secondary structure models of group I twintrons, as well as an example of in vitro ribozyme activity, are presented. We suggest that the group I twintrons have been independently established in myxomycetes and schizopyrenid amoeboflagellates by horizontal gene transfer due to a combination of the phagocytotic behavior in natural environments and the extrachromosomal multicopy nature of ribosomal DNA.

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.

Group I-like ribozymes with a novel core organization perform obligate sequential hydrolytic cleavages at two processing sites.

A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing-ribozyme and a small internal group I-like ribozyme (GIR1), probably involved in protein expression. GIR1 was found to cleave at two internal sites in an obligate sequential order. Both sites are located 3' of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160-190 nt, they are the smallest functional group I ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates a compactly folded ribozyme with the functionally essential P15 exposed in the cleft between the two principal domains P3-P8 and P4-P6.

Two group I ribozymes with different functions in a nuclear rDNA intron.

DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI.