Search

MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs and are implicated in an increasing number of biological processes. To examine whether miRNAs might contribute to sexual differentiation, we performed expression profiling of miRNAs in mouse embryonic gonads with the use of a highly sensitive cloning method, mRAP. Our profiling data revealed substantial differences in the miRNA repertoire between male and female gonads at embryonic (E) day 13.5 (E13.5), suggesting that such differentially expressed miRNAs may function in sexual differentiation. Female-specific miRNAs included miR-29b, which also has been known to be expressed in a similar sex-dependent manner in the gonads of chicken embryos, suggestive of a conserved role in gonadogenesis. Transcripts of the human genes for the de novo methyltransferases DNMT3A and DNMT3B have been identified as targets of miR-29b, and we found that mouse miR-29b also negatively regulates Dnmt3a and Dnmt3b expression in luciferase reporter assays. We also found that miR-29b is expressed in mouse primordial germ cells (PGCs) at E13.5 and that its expression is up-regulated in a female-specific manner between E13.5 and E17.5, when male-specific de novo methylation of the PGC genome is known to occur. Our data thus suggest that miR-29b may play an important role in female gonadal development by targeting Dnmt3a and Dnmt3b and thereby modulating methylation of genomic DNA in PGCs., MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs and are implicated in an increasing number of biological processes. To examine whether miRNAs might contribute to sexual differentiation, we performed expression profiling of miRNAs in mouse embryonic gonads with the use of a highly sensitive cloning method, mRAP. Our profiling data revealed substantial differences in the miRNA repertoire between male and female gonads at embryonic (E) day 13.5 (E13.5), suggesting that such differentially expressed miRNAs may function in sexual differentiation. Female-specific miRNAs included miR-29b, which also has been known to be expressed in a similar sex-dependent manner in the gonads of chicken embryos, suggestive of a conserved role in gonadogenesis. Transcripts of the human genes for the de novo methyltransferases DNMT3A and DNMT3B have been identified as targets of miR-29b, and we found that mouse miR-29b also negatively regulates Dnmt3a and Dnmt3b expression in luciferase reporter assays. We also found that miR-29b is expressed in mouse primordial germ cells (PGCs) at E13.5 and that its expression is up-regulated in a female-specific manner between E13.5 and E17.5, when male-specific de novo methylation of the PGC genome is known to occur. Our data thus suggest that miR-29b may play an important role in female gonadal development by targeting Dnmt3a and Dnmt3b and thereby modulating methylation of genomic DNA in PGCs.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77,436 Small-RNA species was sequenced, with 11,776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner., MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77,436 Small-RNA species was sequenced, with 11,776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.

MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted., MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.