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Identification and characterization of new miRNAs cloned from normal mouse mammary gland.

Sdassi N, Silveri L, Laubier J, Tilly G, Costa J, Layani S, Vilotte JL, Le Provost F - BMC Genomics (2009)

Bottom Line: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes.Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them.If none of them is mammary gland specific, a few of them are not ubiquitously expressed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR 339, Laboratoire de Génétique Biochimique et Cytogénétique, Jouy-en-Josas, France. Nezha.Sdassi@jouy.inra.fr

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.

Results: To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.

Conclusion: The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.

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Chromosomal distribution of the miRNA genes identified in this study. The number of hits represents the number of miRNA genes localized on each chromosome. Thus study did not reveal the occurrence of a specific chromosome encompassing most of the mammary-expressed miRNA loci.
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Figure 2: Chromosomal distribution of the miRNA genes identified in this study. The number of hits represents the number of miRNA genes localized on each chromosome. Thus study did not reveal the occurrence of a specific chromosome encompassing most of the mammary-expressed miRNA loci.

Mentions: Their chromosomal locations and sequence alignments revealed that several cloned sequences were partially overlapping and could derive from the same precursor. In our study 5 families, corresponding to 13 sequences, were detected (Figure 1). Recently, the presence of variants has been reported after the characterization of cDNA libraries of small RNAs from porcine fibroblast cells [35] and from bovine adipose tissue and mammary gland [29]. The 3'-end variants may thus arise from preferential degradation at the 3' end or from imprecise processing of miRNA precursors by Dicer, thereby generating miRNAs with differing 3' ends [36,37]. However, we cannot exclude the possibility that these miRNA variants originate from multiple genomic loci. The functional specialization of miRNA variants is still unknown. The 33 cloned sequences could derive from 41 precursors (Additional file 2). In fact one cloned sequence could derive from several precursors (Additional file 2, for example MG141 could be issue from 3 precursors). In some cases one cloned sequence possess i) 2 identical precursors localised in 2 different chromosomes (Additional file 2, MG016-13 and -16), ii) 3 identical precursors clustered in one chromosome (MG055) or iii) 2 similar precursors (with 1 or 3 different nt) in two different chromosomes (Additional file 2, MG009/MG037/MG056/MG066-06 and -17, MG016-04 and -1 or MG141-12 and -15, for example). Overall the precursors are distributed on all the chromosomes but chromosomes 5, 10 and Y (Figure 2). Chromosome 1 appears to contain more mammary gland miRNA genes than any other chromosome. Our result are in agreement with published results [38,39] that showed that miRNA genes are distributed among all the chromosomes but chromosome Y. Twenty-six of the precursors are localised in intergenic regions, 14 in genes (intron: 11, 5'UTR: 1, 3'UTR: 1 and exon: 1) and 1 corresponds to one miRNA non-fully characterised (ENSMUSG00000076325) (Additional file 2). Thus the majority of miRNA genes are part of intergenic sequences (62%) as observed by Ro et al. [40]. By analysing the proximity of the 57 chromosomal locations in the mouse genome, 4 miRNA clusters were observed (Table 1). The miRNA genes are in the same cluster if they are less than 1000 bp apart on the same chromosome [29]. This physical proximity is consistent with recent reports of miRNA clustering within the human genome [41]. Among the 4 clusters, 2 correspond to the association of two new precursors, MG016-01 and MG016-16, with 2 miRNAs already described mmu-miR-689-1 and mmu-miR-689-2, respectively (Table 1). The cluster MG055 on chromosome 1 has three repeats of the same precursor.


Identification and characterization of new miRNAs cloned from normal mouse mammary gland.

Sdassi N, Silveri L, Laubier J, Tilly G, Costa J, Layani S, Vilotte JL, Le Provost F - BMC Genomics (2009)

Chromosomal distribution of the miRNA genes identified in this study. The number of hits represents the number of miRNA genes localized on each chromosome. Thus study did not reveal the occurrence of a specific chromosome encompassing most of the mammary-expressed miRNA loci.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683868&req=5

Figure 2: Chromosomal distribution of the miRNA genes identified in this study. The number of hits represents the number of miRNA genes localized on each chromosome. Thus study did not reveal the occurrence of a specific chromosome encompassing most of the mammary-expressed miRNA loci.
Mentions: Their chromosomal locations and sequence alignments revealed that several cloned sequences were partially overlapping and could derive from the same precursor. In our study 5 families, corresponding to 13 sequences, were detected (Figure 1). Recently, the presence of variants has been reported after the characterization of cDNA libraries of small RNAs from porcine fibroblast cells [35] and from bovine adipose tissue and mammary gland [29]. The 3'-end variants may thus arise from preferential degradation at the 3' end or from imprecise processing of miRNA precursors by Dicer, thereby generating miRNAs with differing 3' ends [36,37]. However, we cannot exclude the possibility that these miRNA variants originate from multiple genomic loci. The functional specialization of miRNA variants is still unknown. The 33 cloned sequences could derive from 41 precursors (Additional file 2). In fact one cloned sequence could derive from several precursors (Additional file 2, for example MG141 could be issue from 3 precursors). In some cases one cloned sequence possess i) 2 identical precursors localised in 2 different chromosomes (Additional file 2, MG016-13 and -16), ii) 3 identical precursors clustered in one chromosome (MG055) or iii) 2 similar precursors (with 1 or 3 different nt) in two different chromosomes (Additional file 2, MG009/MG037/MG056/MG066-06 and -17, MG016-04 and -1 or MG141-12 and -15, for example). Overall the precursors are distributed on all the chromosomes but chromosomes 5, 10 and Y (Figure 2). Chromosome 1 appears to contain more mammary gland miRNA genes than any other chromosome. Our result are in agreement with published results [38,39] that showed that miRNA genes are distributed among all the chromosomes but chromosome Y. Twenty-six of the precursors are localised in intergenic regions, 14 in genes (intron: 11, 5'UTR: 1, 3'UTR: 1 and exon: 1) and 1 corresponds to one miRNA non-fully characterised (ENSMUSG00000076325) (Additional file 2). Thus the majority of miRNA genes are part of intergenic sequences (62%) as observed by Ro et al. [40]. By analysing the proximity of the 57 chromosomal locations in the mouse genome, 4 miRNA clusters were observed (Table 1). The miRNA genes are in the same cluster if they are less than 1000 bp apart on the same chromosome [29]. This physical proximity is consistent with recent reports of miRNA clustering within the human genome [41]. Among the 4 clusters, 2 correspond to the association of two new precursors, MG016-01 and MG016-16, with 2 miRNAs already described mmu-miR-689-1 and mmu-miR-689-2, respectively (Table 1). The cluster MG055 on chromosome 1 has three repeats of the same precursor.

Bottom Line: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes.Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them.If none of them is mammary gland specific, a few of them are not ubiquitously expressed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR 339, Laboratoire de Génétique Biochimique et Cytogénétique, Jouy-en-Josas, France. Nezha.Sdassi@jouy.inra.fr

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.

Results: To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.

Conclusion: The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.

Show MeSH
Related in: MedlinePlus