List of differentially expressed small RNAs between male and female blastocysts (2).

†<p>Small RNAs showing more than a 10-fold (1000%) change in sequence reads are listed. Small RNAs showing fewer than 20 reads have been excluded from this list.</p

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

<div><p>Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed <i>Xist</i>, an imprinted gene, has been considered as a single <i>cis</i>-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than <i>Xist</i> expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to <i>Xist</i> that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: <i>Jpx</i>, <i>Ftx</i> and <i>Zcchc13</i>. To our knowledge, this is the first identified cluster of imprinted genes in the <i>cis</i>-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.</p></div

Identification of a cluster of imprinted genes in the X-inactivation center (Xic).

<p>A newly identified cluster of imprinted genes on the X chromosome is shown in the mouse genome (UCSC mm9; <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). This cluster is located within the Xic. The arrows show the direction of each transcription unit. <i>Ftx</i> and Z<i>cchc13</i> are transcribed in opposite directions from the same putative bidirectional promoter. Imprinted genes are shown by the thick black arrows; <i>Xist, Jpx, Ftx, Zcchc13</i>, miR-374-5p and miR-421-3p were paternally expressed, while <i>Tsix</i> was reported previously to be maternally expressed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Lee1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Sado1" target="_blank">[29]</a>. The thick black bar indicates the position of the Tg53 (460 kb) transgene reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Okamoto2" target="_blank">[35]</a>.</p

Identification of small RNAs predominantly expressed in female blastocysts.

<p>(A) Scheme of the X^GFP sexing system and construction of small RNA libraries. Transgenic male mice (X^GFPY) were mated with wild-type female mice (XX). Only the female embryos (XX^GFP) fluoresce green because of the paternally inherited X^GFP. Small RNAs obtained from male and female blastocysts were sequenced and expression levels were compared between sexes. (B) Annotation of the read sequences from male and female blastocyst libraries. The distribution of the RNA classes is given as a percentage of the total annotated ncRNAs. (C) Expression levels of miR-374-5p, miR-421-3p and miR-295 miRNA in male and female blastocysts. The expression of three miRNAs was measured by qRT–PCR and normalized to that of snoRNA202 examined as a control. The data indicate that the miR-374-5p and miR-421-3p were predominantly expressed in female blastocysts (*<i>P</i><0.01). Results are expressed as the mean ± SD (<i>n</i> = 3).</p

List of differentially expressed small RNAs between male and female blastocysts (1).

*<p>Sequence reads of small RNAs that showed 0 reads in male embryos and more than 10 reads in female embryos.</p>**<p>Small RNAs showing more than a 10-fold (1000%) change in sequence reads are listed. Small RNAs showing fewer than 20 reads have been excluded from this list.</p

Classification of annotated small RNAs in male and female blastocysts.

*<p>The number of genes mapped is not always equal to the “Total ncRNA” plus “Only genomic” counts because of multiple annotations for single read sequences.</p><p>Abbreviations: miRNA_mature, micro RNA mature form; Mt_tRNA, mitochondrial transfer RNA; Mt_rRNA, mitochondrial ribosomal RNA; snRNA, small nuclear RNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; miscRNA, miscellaneous RNA; lincRNA, large intergenic noncoding RNA; miRNA_hairpin: micro RNA stem-loop sequence obtained from miRBase.</p>**<p>The sequences classified as “Only genomic” were further annotated against RepeatMasker and the tRNA database. The results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222.s002" target="_blank">Table S1</a>.</p

Verification of imprinting of two miRNAs predominantly expressed in female embryos.

<p>(A) Schematic representation of two mature miRNAs, miR-374-5p and miR-421-3p and their preceding transcript pri-miRNA. Both miRNAs were clustered and located in the intron of the <i>Ftx</i> (B230206F22Rik) gene on the X chromosome. Both are thought to be transcribed as pri-miRNAs and then processed to form mature miRNAs. The location of the primers used to assess the pri-miRNA and <i>Ftx</i> expression levels are indicated: qRT–PCR primers 207 and 208 were used for <i>Ftx</i>; primers used for allelic discrimination were 057 and 058 for pri-miRNA, 116 and 117 for <i>Ftx</i>. The exons for <i>Ftx</i> are numbered according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Chureau1" target="_blank">[22]</a>. DNA polymorphisms used for allelic discrimination are indicated with red characters. PCR primers correspond to the list in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222.s004" target="_blank">Table S3</a> (B) Production of mature miRNAs from pre-miRNAs in female blastocysts. Although two mature forms of miRNA, miR-374-5p/miR-374-3p and miR-421-5p/miR-421-3p are registered in miRBase, only miR-374-5p and miR-421-3p were detected at the blastocyst stage. Orange coloring shows mismatch sequences compared with the reference sequences, suggesting miRNA editing or misreading by the 454 sequencer. (C) Expression of pri-miRNA in wild-type male and female blastocysts obtained from the uterus. Female-predominant expression was observed as well as previously reported X-linked imprinted genes such as <i>Xist and Rhox5</i>. (D) Verification of imprinting of the pri-miRNA for miR-374-5p and miR-421-3p in preimplantation mouse embryos. Pri-miRNA was expressed from the paternal B6 allele in JF1× C57BL/6 crosses and from the paternal JF1 allele in the reciprocal B6× (JF1× B6) cross. The arrows indicate the polymorphism sites that were used in this assay.</p

Distribution of the predicted insert size in the second version of the full-length cDNA library (PnFL2)

<p><b>Copyright information:</b></p><p>Taken from "Functional annotation of 19,841 full-length enriched cDNA clones"</p><p>http://www.biomedcentral.com/1471-2164/8/448</p><p>BMC Genomics 2007;8():448-448.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222646.</p><p></p> The fragment sizes of 17,273 CDS that were substituted for the ESTs were determined

The putative physical distribution of PnFL clones in 19 chromosomes (top: north; bottom: south)

<p><b>Copyright information:</b></p><p>Taken from "Functional annotation of 19,841 full-length enriched cDNA clones"</p><p>http://www.biomedcentral.com/1471-2164/8/448</p><p>BMC Genomics 2007;8():448-448.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222646.</p><p></p> Each red pixel shows a locus that corresponds to a PnFL clone. The number underneath each chromosome is the chromosome number and that above is the distribution index (see )

Mouse Model for ROS1-Rearranged Lung Cancer

<div><p>Genetic rearrangement of the <em>ROS1</em> receptor tyrosine kinase was recently identified as a distinct molecular signature for human non-small cell lung cancer (NSCLC). However, direct evidence of lung carcinogenesis induced by <em>ROS1</em> fusion genes remains to be verified. The present study shows that <em>EZR-ROS1</em> plays an essential role in the oncogenesis of NSCLC harboring the fusion gene. <em>EZR-ROS1</em> was identified in four female patients of lung adenocarcinoma. Three of them were never smokers. Interstitial deletion of 6q22–q25 resulted in gene fusion. Expression of the fusion kinase in NIH3T3 cells induced anchorage-independent growth <em>in vitro</em>, and subcutaneous tumors in nude mice. This transforming ability was attributable to its kinase activity. The ALK/MET/ROS1 kinase inhibitor, crizotinib, suppressed fusion-induced anchorage-independent growth of NIH3T3 cells. Most importantly, established transgenic mouse lines specifically expressing EZR-ROS1 in lung alveolar epithelial cells developed multiple adenocarcinoma nodules in both lungs at an early age. These data suggest that the <em>EZR-ROS1</em> is a pivotal oncogene in human NSCLC, and that this animal model could be valuable for exploring therapeutic agents against <em>ROS1</em>-rearranged lung cancer.</p> </div