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Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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Characterization of pancRNAs by directional RNA-seq. (A) Screening of pancRNA datasets. One hundred oocytes or 2-cell embryos were used for each analysis, and four replicates were made for the statistical tests. (B) Numbers of pancRNA and mRNA species present in 2-cell embryos. (C) Violin plot of mRNA levels of 2-cell embryo genes with and without pancRNAs. Groups A, B and C comprise mRNAs without pancRNAs (11658), with the 100 most weakly expressed pancRNAs, and with the 100 most strongly expressed pancRNAs, respectively. Violin width and white circles indicate gene density and median expression levels of mRNAs, respectively. Box plots were merged and are indicated by black bars. ***P<0.001. (D) Expression difference of the pancRNA partners of the upregulated genes and their probability visualized as a density plot. Twofold upregulated genes (which correspond to 520 of the 836 genes in B) were selected. Below the density plot is a unidimensional plot of the expression difference of each pancRNA (circles), in which density is expressed by color intensity. (E) A frequently observed sequence motif in the promoter regions of pancRNA-partnered genes. (F) The frequency of the sequence motif in various gene regions. Sequences with 90% or greater identity to the position weight matrix (pwm) of the motif shown in E were categorized according to their presence on the sense or antisense strand of the promoter (−500 to −1 bp) and gene body (+1 to +500 bp) regions. The TSSs of the pancRNA-partnered genes provide the switching points for the observed asymmetric distribution of the CT-rich sequence.
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DEV116996F1: Characterization of pancRNAs by directional RNA-seq. (A) Screening of pancRNA datasets. One hundred oocytes or 2-cell embryos were used for each analysis, and four replicates were made for the statistical tests. (B) Numbers of pancRNA and mRNA species present in 2-cell embryos. (C) Violin plot of mRNA levels of 2-cell embryo genes with and without pancRNAs. Groups A, B and C comprise mRNAs without pancRNAs (11658), with the 100 most weakly expressed pancRNAs, and with the 100 most strongly expressed pancRNAs, respectively. Violin width and white circles indicate gene density and median expression levels of mRNAs, respectively. Box plots were merged and are indicated by black bars. ***P<0.001. (D) Expression difference of the pancRNA partners of the upregulated genes and their probability visualized as a density plot. Twofold upregulated genes (which correspond to 520 of the 836 genes in B) were selected. Below the density plot is a unidimensional plot of the expression difference of each pancRNA (circles), in which density is expressed by color intensity. (E) A frequently observed sequence motif in the promoter regions of pancRNA-partnered genes. (F) The frequency of the sequence motif in various gene regions. Sequences with 90% or greater identity to the position weight matrix (pwm) of the motif shown in E were categorized according to their presence on the sense or antisense strand of the promoter (−500 to −1 bp) and gene body (+1 to +500 bp) regions. The TSSs of the pancRNA-partnered genes provide the switching points for the observed asymmetric distribution of the CT-rich sequence.

Mentions: To examine whether pancRNAs are induced after fertilization, we generated a total of 111 million directional RNA-seq reads using an Illumina HiSeq2000 from mouse metaphase II (MII) oocytes and 420 million directional RNA-seq reads from 2-cell embryos (see Materials and Methods). These reads were mapped to the mouse mm10 genome. Our RNA-seq data showed robust reproducibility among biological replicates (Pearson correlation coefficient, r>0.99; supplementary material Fig. S1). 5′-3′ mapping bias was comparable to that of RNA-seq data for oocytes in previous studies (Park et al., 2013) (supplementary material Fig. S2). In order to verify the strandedness of our directional RNA-seq data, we mapped the reads to known RefSeq genes and calculated the proportion that mapped on the correct strand. The results showed that 99.1% of the reads from MII oocytes and 96.9% of the reads from the fertilized 2-cell embryos mapped on the correct strand. Using our high-resolution datasets, we identified 618 and 1129 candidate pancRNAs in MII oocytes and 2-cell embryos, respectively (Fig. 1A).Fig. 1.


Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Characterization of pancRNAs by directional RNA-seq. (A) Screening of pancRNA datasets. One hundred oocytes or 2-cell embryos were used for each analysis, and four replicates were made for the statistical tests. (B) Numbers of pancRNA and mRNA species present in 2-cell embryos. (C) Violin plot of mRNA levels of 2-cell embryo genes with and without pancRNAs. Groups A, B and C comprise mRNAs without pancRNAs (11658), with the 100 most weakly expressed pancRNAs, and with the 100 most strongly expressed pancRNAs, respectively. Violin width and white circles indicate gene density and median expression levels of mRNAs, respectively. Box plots were merged and are indicated by black bars. ***P<0.001. (D) Expression difference of the pancRNA partners of the upregulated genes and their probability visualized as a density plot. Twofold upregulated genes (which correspond to 520 of the 836 genes in B) were selected. Below the density plot is a unidimensional plot of the expression difference of each pancRNA (circles), in which density is expressed by color intensity. (E) A frequently observed sequence motif in the promoter regions of pancRNA-partnered genes. (F) The frequency of the sequence motif in various gene regions. Sequences with 90% or greater identity to the position weight matrix (pwm) of the motif shown in E were categorized according to their presence on the sense or antisense strand of the promoter (−500 to −1 bp) and gene body (+1 to +500 bp) regions. The TSSs of the pancRNA-partnered genes provide the switching points for the observed asymmetric distribution of the CT-rich sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352986&req=5

DEV116996F1: Characterization of pancRNAs by directional RNA-seq. (A) Screening of pancRNA datasets. One hundred oocytes or 2-cell embryos were used for each analysis, and four replicates were made for the statistical tests. (B) Numbers of pancRNA and mRNA species present in 2-cell embryos. (C) Violin plot of mRNA levels of 2-cell embryo genes with and without pancRNAs. Groups A, B and C comprise mRNAs without pancRNAs (11658), with the 100 most weakly expressed pancRNAs, and with the 100 most strongly expressed pancRNAs, respectively. Violin width and white circles indicate gene density and median expression levels of mRNAs, respectively. Box plots were merged and are indicated by black bars. ***P<0.001. (D) Expression difference of the pancRNA partners of the upregulated genes and their probability visualized as a density plot. Twofold upregulated genes (which correspond to 520 of the 836 genes in B) were selected. Below the density plot is a unidimensional plot of the expression difference of each pancRNA (circles), in which density is expressed by color intensity. (E) A frequently observed sequence motif in the promoter regions of pancRNA-partnered genes. (F) The frequency of the sequence motif in various gene regions. Sequences with 90% or greater identity to the position weight matrix (pwm) of the motif shown in E were categorized according to their presence on the sense or antisense strand of the promoter (−500 to −1 bp) and gene body (+1 to +500 bp) regions. The TSSs of the pancRNA-partnered genes provide the switching points for the observed asymmetric distribution of the CT-rich sequence.
Mentions: To examine whether pancRNAs are induced after fertilization, we generated a total of 111 million directional RNA-seq reads using an Illumina HiSeq2000 from mouse metaphase II (MII) oocytes and 420 million directional RNA-seq reads from 2-cell embryos (see Materials and Methods). These reads were mapped to the mouse mm10 genome. Our RNA-seq data showed robust reproducibility among biological replicates (Pearson correlation coefficient, r>0.99; supplementary material Fig. S1). 5′-3′ mapping bias was comparable to that of RNA-seq data for oocytes in previous studies (Park et al., 2013) (supplementary material Fig. S2). In order to verify the strandedness of our directional RNA-seq data, we mapped the reads to known RefSeq genes and calculated the proportion that mapped on the correct strand. The results showed that 99.1% of the reads from MII oocytes and 96.9% of the reads from the fertilized 2-cell embryos mapped on the correct strand. Using our high-resolution datasets, we identified 618 and 1129 candidate pancRNAs in MII oocytes and 2-cell embryos, respectively (Fig. 1A).Fig. 1.

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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