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New miRNAs cloned from neuroblastoma.

Afanasyeva EA, Hotz-Wagenblatt A, Glatting KH, Westermann F - BMC Genomics (2008)

Bottom Line: Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting.Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes.We provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Tumour Genetics, B030, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. e.afanasyeva@dkfz-heidelberg.de

ABSTRACT

Background: MicroRNAs (miRNAs) are a novel class of gene expression regulators implicated in cancer biology. Neuroblastoma (NB) is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. Recent advances identified a subset of miRNAs with putative function in NB biology. However, the full repertoire of miRNAs expressed in NBs is not available.

Results: We describe miRNA profiles of 13 NB specimens and 2 NB cell lines as determined by miRNA cloning. A total of 3153 sequences were sequenced and analysed by a miRNA prediction tool (miRpredict). Our library covered 27% miRNAs known to date. 39 reads corresponding to 25 individual sequences were classified as novel miRNAs, including miRNA* species of 10 known miRNAs. Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting. Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes.

Conclusion: We provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species). Some of these newly identified sequences reside within frequently altered chromosomal regions in NB tumours and may play a role in NB biology.

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MYCNAMP_NB4_70, a novel miRNA-like sequence resulting from a duplication event. Short sequences flanking MYCNAMP_NB4_70 were extracted from the human genome, as well as homologuous sequences from chimpanzee, macaque, mouse, rat and dog genomes and aligned. The bar indicates the MYCNAMP_NB4_70 sequence. The duplication is marked by a box.
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Figure 1: MYCNAMP_NB4_70, a novel miRNA-like sequence resulting from a duplication event. Short sequences flanking MYCNAMP_NB4_70 were extracted from the human genome, as well as homologuous sequences from chimpanzee, macaque, mouse, rat and dog genomes and aligned. The bar indicates the MYCNAMP_NB4_70 sequence. The duplication is marked by a box.

Mentions: Other 15 sequences represent novel small RNAs which have not been annotated. Two novel sequences were found adjacent to chromosomal localisations of known miRNAs: MYCNSC_NB5_330 within a miRNA cluster on chromosome 14 as well as MYCNAMP_NB2_61 within a miRNA cluster on chromosome 19 (Table 3). Phylogenetic conservation was determined for each putative miRNA and its surrounding with respect to chimpanzee, macaque, opossum, mouse, rat, dog, bull and chicken genomes (see Additional File 2). We found that the cloned sequences with the exception of MYCNAMP_NB4_70 are conserved either within all chosen species or primates. The non-conserved MYCNAMP_NB4_70 sequence seems to be the result of a short duplication within an intron of the DPP10 transcript (Fig. 1). However, homologous regions in chimpanzee and macaque genomes are also able to form hairpins which might give rise to miRNAs. Novel miRNAs do not share any homology with each other and therefore do not comprise a family. Searching against miRBase revealed that none of the new miRNAs are related to the annotated miRNAs in miRBase, except of one sequence, MYCNAMP_NB2_5, that is homologous to hsa-mir-151 and -28. Analysis of genomic locations of the individual miRNAs showed that 5 of them are localized in extragenic regions. 8 sequences are found within introns of coding transcripts. MYCNSC_NB5_64, classified as a miRNA by miRpredict, is found within a predicted U5 gene which reduces reliability of this sequence as a miRNA (Table 3). The likely precursors of the novel miRNAs (Fig. 2) may be subdivided into two subgroups: structures with typical hairpin or borderline precursors. The latter subgroup has features divergent from a canonical miRNA hairpin, such as bulges (KELLY_276, MYCNSC_NB2_148), short stem (MYCNSC_NB5_41) or "oscillating" hairpin (MYCNAMP_NB4_70), where the candidate miRNA can reside on the 5' or 3'-arm. Lui et al. reported the cloning of a subset of non-canonical miRNAs, however the relevance of such RNAs to the classical miRNA pathway remains to be determined [27].


New miRNAs cloned from neuroblastoma.

Afanasyeva EA, Hotz-Wagenblatt A, Glatting KH, Westermann F - BMC Genomics (2008)

MYCNAMP_NB4_70, a novel miRNA-like sequence resulting from a duplication event. Short sequences flanking MYCNAMP_NB4_70 were extracted from the human genome, as well as homologuous sequences from chimpanzee, macaque, mouse, rat and dog genomes and aligned. The bar indicates the MYCNAMP_NB4_70 sequence. The duplication is marked by a box.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254388&req=5

Figure 1: MYCNAMP_NB4_70, a novel miRNA-like sequence resulting from a duplication event. Short sequences flanking MYCNAMP_NB4_70 were extracted from the human genome, as well as homologuous sequences from chimpanzee, macaque, mouse, rat and dog genomes and aligned. The bar indicates the MYCNAMP_NB4_70 sequence. The duplication is marked by a box.
Mentions: Other 15 sequences represent novel small RNAs which have not been annotated. Two novel sequences were found adjacent to chromosomal localisations of known miRNAs: MYCNSC_NB5_330 within a miRNA cluster on chromosome 14 as well as MYCNAMP_NB2_61 within a miRNA cluster on chromosome 19 (Table 3). Phylogenetic conservation was determined for each putative miRNA and its surrounding with respect to chimpanzee, macaque, opossum, mouse, rat, dog, bull and chicken genomes (see Additional File 2). We found that the cloned sequences with the exception of MYCNAMP_NB4_70 are conserved either within all chosen species or primates. The non-conserved MYCNAMP_NB4_70 sequence seems to be the result of a short duplication within an intron of the DPP10 transcript (Fig. 1). However, homologous regions in chimpanzee and macaque genomes are also able to form hairpins which might give rise to miRNAs. Novel miRNAs do not share any homology with each other and therefore do not comprise a family. Searching against miRBase revealed that none of the new miRNAs are related to the annotated miRNAs in miRBase, except of one sequence, MYCNAMP_NB2_5, that is homologous to hsa-mir-151 and -28. Analysis of genomic locations of the individual miRNAs showed that 5 of them are localized in extragenic regions. 8 sequences are found within introns of coding transcripts. MYCNSC_NB5_64, classified as a miRNA by miRpredict, is found within a predicted U5 gene which reduces reliability of this sequence as a miRNA (Table 3). The likely precursors of the novel miRNAs (Fig. 2) may be subdivided into two subgroups: structures with typical hairpin or borderline precursors. The latter subgroup has features divergent from a canonical miRNA hairpin, such as bulges (KELLY_276, MYCNSC_NB2_148), short stem (MYCNSC_NB5_41) or "oscillating" hairpin (MYCNAMP_NB4_70), where the candidate miRNA can reside on the 5' or 3'-arm. Lui et al. reported the cloning of a subset of non-canonical miRNAs, however the relevance of such RNAs to the classical miRNA pathway remains to be determined [27].

Bottom Line: Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting.Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes.We provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Tumour Genetics, B030, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. e.afanasyeva@dkfz-heidelberg.de

ABSTRACT

Background: MicroRNAs (miRNAs) are a novel class of gene expression regulators implicated in cancer biology. Neuroblastoma (NB) is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. Recent advances identified a subset of miRNAs with putative function in NB biology. However, the full repertoire of miRNAs expressed in NBs is not available.

Results: We describe miRNA profiles of 13 NB specimens and 2 NB cell lines as determined by miRNA cloning. A total of 3153 sequences were sequenced and analysed by a miRNA prediction tool (miRpredict). Our library covered 27% miRNAs known to date. 39 reads corresponding to 25 individual sequences were classified as novel miRNAs, including miRNA* species of 10 known miRNAs. Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting. Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes.

Conclusion: We provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species). Some of these newly identified sequences reside within frequently altered chromosomal regions in NB tumours and may play a role in NB biology.

Show MeSH
Related in: MedlinePlus