1 publications mentioning ptr-mir-127

Open access articles that are associated with the species Pan troglodytes and mention the gene name mir-127. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

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Alteration of those miRNAs expression may in turn affect the expression of the imprinted genes, as evidenced by the down-regulation of Rtl1 by miR-127 in this study.

Thus, the expression of human miR-127 was ∼20 times higher than human miR-433, the expression of rat miR-127 was ∼47 times higher than rat miR-433, and the expression of dog miR-127 was ∼40 times higher than dog miR-433.

Down-Regulation of the Imprinted Gene Rtl1 by Overexpression of miR-127.

This data suggests that miR-127 may function as a siRNA to down-regulate its host gene expression.

Potential PCR amplification from genomic DNA contamination was eliminated by treating the total RNA with DNase I. As shown in Figures 6a–c, primary transcripts of the human (a), rat (b), or dog (c) miR-433 and miR-127 were easily detected in cells that over-expressed the recombinant expression vector, respectively.

0007829.g005 Figure 5(a) The miR-433/127 loci expression plasmid of human, rat, or dog was expressed in mouse Hepa-1 cells (different species), and the binding of ERRγ to the endogenous promoter of miR-433 and of miR-127 of each species was detected using specific ERRγ antibodies.

0007829.g006 Figure 6(Panels a–c) Semi-quantitative RT-PCR analysis of pri-miR-433 and pri-miR-127 expression transcribed from the human (a), rat (b), and dog (c) miR-433/127 loci expression plasmid.

The recombinant human, rat, or dog miR-433/127 loci expression vector (designated pMIR-REPORT-NoMp-human, rat or dog) was then transfected into Hepa-1 cells and the expression of miR-433 and miR-127 primary transcripts was examined using semi-quantitative RT-PCR.

Because miR-127 is located in an imprinted region encoding Rtl1, we determined if changes in miR-127 expression would affect the expression of Rtl1.

Artificial Expression of miR-433 and miR-127 In Vitro in CellsOur previous studies showed that the mouse miR-433 and miR-127 genes overlapped and the expression of mouse miR-433 and miR-127 was controlled by independent promoters [3], [4].

0007829.g007 Figure 7 The expression vector of miR-127 was transfected in human Hela and mouse Hepa-1 cells and the expression of Rtl1 was determined by a modified strand-specific q-PCR.

Similarly, the rat miR-433 and miR-127 was ∼210 fold and ∼10000 fold ovexpressed (Figure 6f) and the dog miR-433 and miR-127 was ∼4 fold and ∼160 fold ovexpressed (Figure 6g), respectively.

The expression vector of miR-127 was transfected in human Hela and mouse Hepa-1 cells and the expression of Rtl1 was determined by a modified strand-specific q-PCR.

We overexpressed miR-127 in human Hela and mouse Hepa-1 cells using an expression vector of miR-127 and the level of Rtl1 was assessed using a strand specific q-PCR analysis.

For instance, if mutations occurred in the region between pre-miR-433 and pre-miR-127, it would be likely to affect both the processing of mature miR-433 and the regulation of miR-127 expression.

We next determined expression levels of the mature miR-433 and miR-127 in Hepa-1 cells after recombinant vector over -expression.

Interestingly, ectopic expression of miR-127 resulted in a significant reduction in Rtl1 expression in both cells (Figure 7).

Based on this information, we made a direct comparison between the expression level of miR-433 and miR-127.

Compared with non-vector transfected cells, the human miR-433 was ∼100 fold over-expressed whereas the human miR-127 was ∼2000 fold over-expressed (Figure 6e).

The imprinted expression of miR-127 has been shown to undergo DNA methylation regulation in mouse embryos [26] and cancer cells [27].

Our studies provide evidence for a conserved gene structure and ERR/SHP dependent regulation of miR-433 and miR-127 gene expression in mammals.

We recently have shown that gene expression of miR-433 and miR-127 in mice was regulated via a nuclear receptor ERRγ/SHP dependent mechanism [4].

Based on the identification of the same binding motifs, we predicted that a common regulatory mechanism of miR-433 and miR-127 expression may exist among different mammalian species.

Artificial Expression of miR-433 and miR-127 In Vitro in Cells.

Due to high expression of primary transcript of miR-433 and mIR-127, different PCR cycles were used (see Figure 6).

This would allow us to determine if the transcriptional expression of miR-433 or miR-127 could be driven by its own promoter from the endogenous miR-433/127 loci.

The mouse cell line Hepa1 and human cell line Hela were transfected with miR-127 expression vector.

On the other hand, we observed a common transcriptional mechanism governing the expression of miR-433 and miR-127 in mammals, which involved ERR family members and orphan receptor SHP.

As expected, ERRγ dose -dependently activated promoters of miR-433 and miR-127 of human (Figure 4a), rat (Figure 4b), and dog (Figure 4c), which was repressed by co -expression of SHP.

Transcriptional expression of miR-433 and miR-127 from the miR-433/127 loci in human, rat, and dog.

In addition, the expression level of pri-miR-433 was markedly lower than that of pri-miR-127 (Figure 6d) in all three species, suggesting pri-miR-433 and pri-miR-127 were transcribed differentially from an independent transcription unit.

These differential expression results provided further evidence that miR-433 and miR-127 produced from the miR-433-127 loci were transcribed from two separate promoters in human, rat, and dog.

Our previous studies showed that the mouse miR-433 and miR-127 genes overlapped and the expression of mouse miR-433 and miR-127 was controlled by independent promoters [3], [4].

In order to avoid the effect of endogenous miR-127 and miR-433 primary transcripts in Hela cells, we overexpressed the pMIR-Report human433/127 loci, pMIR-Report rat433/127 loci, and pMIR-Report dog433/127 loci in Hepa1 cells.

The expression level of primary transcript of miR-433 and miR-127 was determined by semi-quantitative PCR using primers listed in Supplementary Material Table S3.

We recently have reported that the full length primary transcripts of mouse miR-433 and miR-127 overlapped in a 5′–3′ unidirectional way [3].

We elucidated a common regulatory mechanism governing miR-433 and miR-127 promoter activities, which was dependent on nuclear receptor estrogen related receptor gamma (ERRγ, NR3B3) and small heterodimer partner (SHP, NR0B2) [4].

In this study, we used pMIR-Report-human 433/127, pMIR-Report-rat433/127, pMIR-Report-dog433/127 plasmids as the standard template to determine the PCR efficiency, then compared the expression level of miR-433 and miR-127 primary transcripts.

The conservation of the transcriptional regulation of miR-433 and miR-127 further supports the notion that the miR-433/127 loci in mammals might be evolved from an ancient common origin.

Primers used to determine the expression of primary transcripts of miR-433 and of miR-127 are located surrounding the precursors of miR-433 and of miR-127.

In conclusion, our studies for the first time provide evidence for a conserved structure and transcriptional regulation of the clustered miR-433 and miR-127 genes in mammals, including humans.

However, the question remains to be determined whether molecular details of miR-433 and miR-127 regulation by ERR/SHP are restricted to mouse or whether they apply to other species.

Common Regulation of miR-433 and miR-127 Promoter Activity among Mammalian Species.

Our published results showed that miR-433 and miR-127 genes are overlapped in a 5′–3′ unidirectional way in mouse [3] and these two non-coding genes have an antisense transcript, RTL1 [19].

Based on their overlapping gene structure and transcriptional initiation and termination sites, we subsequently cloned promoters of miR-433 and miR-127.

ERRγ was co-immunoprecipitated on the ERRE containing the endogenous promoter regions of miR-433 and miR-127 in the liver of human and rat, and dog spleen, respectively (Figure 5b).

Although the miR-433/127 loci were located on different chromosomes (Chr) in those species (human, Chr 14; Chimpanzee, Chr14; Horse, Chr 24; Dog, Chr 8; Monkey, Chr 7; Rat, Chr 6; Cow, Chr 21; mouse, Chr 12), multiple sequence alignment (MSA) of the precursors, pre-miR-433 and pre-miR-127, showed that the sequence similarity of pre-miR-433 hairpins was ∼95% (Figure 1a) and of pre-miR-127 was 100% (Figure 1b) among those species.

To determine if miR-433 and miR-127 in human, rat, and dog can be independently and differentially transcribed using each miRNA's own promoter, we cloned a large (∼4.5 kb) human, rat, or dog genomic DNA fragment containing miR-433 and miR-127 and their promoter regions into pMIR-REPORT vector (Figure S2).

Figure S1 Transient transfection assays to determine ERRα and ERRβ regulation of miR-433 and miR-127 promoter (pro. )

Transient transfection assays to determine ERRγ and SHP regulation of miR-433 and miR-127 promoter (pro. )

MatInspector of Genomatix Software Suite was used to predict the transcription factor binding sites in the promoter regions of miR-433 and miR-127 in different species, which was completely using Default parameters (http://www.

0007829.g004 Figure 4Transient transfection assays to determine ERRγ and SHP regulation of miR-433 and miR-127 promoter (pro. )

The mature sequences of miR-433 and miR-127 were identical among the eight species.

The response elements of the miR-433 and miR-127 genes from eight different species are illustrated.

As shown in Figure 5, ERRγ was found to Co-IP on both the miR-433 and miR-127 promoters of human, rat, or dog.

This resulted in the cloning of the gene cluster encoding mouse miR-433 and miR-127, which provided the first report for an overlapping code usage of the paired miR-433/127 gene [3].

The promoters of pri-miR-433 and pri-miR-127 were cloned into a pGL3-basic vector, respectively.

ERRa, but not ERRβ, showed strong activation of miR-433 and miR-127 promoters of human, rat, and dog (Figure S1).

The distance between miR-433 and miR-127 showed a striking similarity: 986 bp in human, chimpanzee, horse, dog, and monkey, 989 bp in rat, 988 bp in cow, and 1007 bp in mouse.

The promoter of pri-miR-433 and of pri-miR-127 from each species was cloned into a pGL3-basic vector, respectively.

Our results presented in this study provide evidence that the miR-433 and miR-127 overlapping genes have a higher rate of conservation in mammalian species.

The genomic region between the two pre-miRNAs is predicted to function as the promoter of miR-127 based on our published mouse data [4].

BLASTN search of genome sequences of different species was completed online and a 5 kb genomic sequence surrounding the miR-433 and miR-127 precursors in each species was extracted manually.

ChIP analysis of ERRγ Co-immunoprecipitation (Co-IP) on the miR-433 and miR-127 promoter region containing putative ERRE in human, rat, and dog.

The precursor sequences of miR-433 and miR-127 were downloaded from the Sanger Institute (http://microrna.

MSA of miR-433 and miR-127 gene promoters in eight mammalian species.

Using mouse miR-433 and miR-127 precursor hairpin structure sequences as a query, we searched the genome databases of seven other species, including human, chimpanzee, horse, dog, monkey, rat, and cow.

The promoters of miR-433 and miR-127 from human, rat and dog were cloned into a pGL3 basic vector, respectively.

Although the miR-433/127 gene loci was located on different chromosomes in different species, the distance between miR-433 and miR-127 is very similar, which is ∼1 kb.

The 4.5 kb genomic sequences centered miR-433 and miR-127 were extracted.

Representative common TF binding motifs (1°∼4°) are shown, and their positions appear to be conserved in the promoter region of miR-433 and of miR-127 among different species.

Predicted ERRE sites on the miR-433 and miR-127 gene promoters.

Promoter analysis of miR-433 and miR-127 luciferase reporters of human, rat and dog.

Why is the miR-433 and miR-127 overlapping gene structure in mammalian species so conserved?

0007829.g003 Figure 3 The response elements of the miR-433 and miR-127 genes from eight different species are illustrated.

Conserved Transcription Factor Binding Motifs in the Upstream Region of miR-433 and of miR-127 among Mammalian Species.

Finally, the long DNA fragments containing human, rat or dog miR-433 and miR-127 loci were inserted into Asc I and Pac I sites of pMIR-REPORT-NoMp.

We used MatInspector of Genomatix Software Suite to identify transcription factor binding motifs and position preference in the upstream promoter regions of miR-433 and of miR-127 in eight mammalian species.

We found that miR-433 and miR-127 had almost identical PCR amplification efficiency (Supplementary Material Table S4).

Conserved response elements in the promoters of miR-433 and miR-127 of eight mammalian species.

Semi-Quantitative RT-PCR for miR-433 and miR-127 Primary Transcripts.

Based on the above gene structure analysis and sequence prediction referenced from our published mouse data [3], [4], we hypothesized that the genomic location of promoters of miR-433 and miR-127 was similar in other mammalian species as in mouse.

In the present study, we analyzed genes encoding miR-433 and miR-127 and determined the promoter transactivation of miR-433 and miR-127 from other mammalian species, including humans.

To further confirm this result, the direct association of ERRγ with miR-433 and miR-127 promoters of each species in vivo was assessed using ChIP assays.

The lowest evolutionary distance between cow and other species is 0.10114 and the sequence homology is low, based on the sequence alignment of the miR-127 promoter region (Figure 2b).

miR-127 pro.

The sequence similarity in either the miR-433 promoter region (Figure 2a) or the genomic region between pre-miR-433 and pre-miR-127 (Figure 2b) was low among the eight species.

We cloned the promoters of miR-433 and miR-127 into pGL3 luciferase reporters using genomic DNAs isolated from liver specimens of human, rat and dog.

The conservation of pre-miR-433 and pre-miR-127 hairpin sequences as well as the distance between them among different mammalian species raised the possibility that miR-433 and miR-127 might be evolved from the same DNA origin during evolution.

Multiple sequence alignment (MSA) of miR-433 and miR-127 precursor hairpin sequences in eight mammalian species.

Despite lower sequence similarity, analysis of miR-433 and miR-127 promoters of those species predicted common nuclear receptor binding sites, including ERRE (Figure 3 and Table 1).

Unique potential binding motifs were also identified in the promoter region of miR-433 and of miR-127 in each species.