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A useful approach to total analysis of RISC-associated RNA.

Hayashida Y, Nishibu T, Inoue K, Kurokawa T - BMC Res Notes (2009)

Bottom Line: After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences.Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates.Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genome Research Laboratories, Wako Pure Chemical Industries, Ltd, Takada 6-1, Amagasaki, Hyogo 661-0963, Japan. hayashida.yukinobu@wako-chem.co.jp

ABSTRACT

Background: Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.

Findings: With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 107 cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

Conclusion: Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.

No MeSH data available.


Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection. Seventeen clones with high score percentile for the miR-122 target site (> = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.
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Figure 5: Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection. Seventeen clones with high score percentile for the miR-122 target site (> = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.

Mentions: We selected 17 mRNA clones with high context score percentile (≧ 40) from the miR-122- and GL3-transfectants, and compared the mRNA content in the immunoprecipitate with that in total RNA by real-time PCR. The difference in the RNA content of individual mRNA is shown in Figure 5. Seven mRNA clones selected only from miR-122 transfectant (Figure 5A) were enriched in RISC of the miR-122-transfectants. And since total RNA amounts of four of these clones, ALDOA (aldolase A, fructose-bisphosphate), PIGS (phosphatidylinositol glycan anchor biosynthesis, class S), SLC1A5 (solute carrier family 1 (neutral amino acid transporter), member 5) and SLC7A5 (solute carrier family 7 (cationic amino acid transporter y+system), member 5) were decreased in the miR-122 transfectants, these mRNAs are thought to be candidates of miR-122 target. These 4 clones is likely to be down-regulated through interaction with miR-122. In fact, in the RefExA microarray database, these 4 mRNAs are expressed at higher levels in HepG2 cells where less miR-122 is expressed than in normal liver with abundant miR-122. With the exception of ALDOA, these mRNAs have not yet been reported as miR-122 targets. To address this issue, other experiments such as translation repression assays may be necessary. Among these candidates, SLC1A5 and SLC7A5 belong to the amino acid transporters and expressions of these transporters are thought to be necessary for the growth of human liver cancer cells [29], and over-expression of SLC1A5 and SLC7A5 in liver cancer cells may correlate with a low miR-122 level.


A useful approach to total analysis of RISC-associated RNA.

Hayashida Y, Nishibu T, Inoue K, Kurokawa T - BMC Res Notes (2009)

Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection. Seventeen clones with high score percentile for the miR-122 target site (> = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748084&req=5

Figure 5: Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection. Seventeen clones with high score percentile for the miR-122 target site (> = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.
Mentions: We selected 17 mRNA clones with high context score percentile (≧ 40) from the miR-122- and GL3-transfectants, and compared the mRNA content in the immunoprecipitate with that in total RNA by real-time PCR. The difference in the RNA content of individual mRNA is shown in Figure 5. Seven mRNA clones selected only from miR-122 transfectant (Figure 5A) were enriched in RISC of the miR-122-transfectants. And since total RNA amounts of four of these clones, ALDOA (aldolase A, fructose-bisphosphate), PIGS (phosphatidylinositol glycan anchor biosynthesis, class S), SLC1A5 (solute carrier family 1 (neutral amino acid transporter), member 5) and SLC7A5 (solute carrier family 7 (cationic amino acid transporter y+system), member 5) were decreased in the miR-122 transfectants, these mRNAs are thought to be candidates of miR-122 target. These 4 clones is likely to be down-regulated through interaction with miR-122. In fact, in the RefExA microarray database, these 4 mRNAs are expressed at higher levels in HepG2 cells where less miR-122 is expressed than in normal liver with abundant miR-122. With the exception of ALDOA, these mRNAs have not yet been reported as miR-122 targets. To address this issue, other experiments such as translation repression assays may be necessary. Among these candidates, SLC1A5 and SLC7A5 belong to the amino acid transporters and expressions of these transporters are thought to be necessary for the growth of human liver cancer cells [29], and over-expression of SLC1A5 and SLC7A5 in liver cancer cells may correlate with a low miR-122 level.

Bottom Line: After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences.Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates.Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genome Research Laboratories, Wako Pure Chemical Industries, Ltd, Takada 6-1, Amagasaki, Hyogo 661-0963, Japan. hayashida.yukinobu@wako-chem.co.jp

ABSTRACT

Background: Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.

Findings: With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 107 cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

Conclusion: Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.

No MeSH data available.