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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.


Categories of cDNA clones derived from immunoprecipitated small RNA of HeLa cells. A: Composition of small RNA cDNA clones. The composition of cDNA clones is shown in a circle graph. Almost all of the recovered cDNA was miRNA (94%), while the remainder was rRNA and tRNA clones (3% and 2%). 'Others' indicates the cDNA clones with hits in the genomic sequence. B: Contents of miRNA clones. Each miRNA clone was deduced by BLAST search. The y-axis indicates number of clones of each miRNA species.
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Figure 1: Categories of cDNA clones derived from immunoprecipitated small RNA of HeLa cells. A: Composition of small RNA cDNA clones. The composition of cDNA clones is shown in a circle graph. Almost all of the recovered cDNA was miRNA (94%), while the remainder was rRNA and tRNA clones (3% and 2%). 'Others' indicates the cDNA clones with hits in the genomic sequence. B: Contents of miRNA clones. Each miRNA clone was deduced by BLAST search. The y-axis indicates number of clones of each miRNA species.

Mentions: The cDNA of the isolated small RNA was synthesized by improved adaptor ligation protocol see Methods section. We utilized the unique features of ThermoPhage™ single-stranded DNA ligase, which has ligase activities for both single-stranded RNA and DNA and can be used at 60°C [23]; this ligase was used for all of the adaptor ligation. High temperature may be advantageous for the ligation of nucleotides with tight tertiary structures. Using this method, cDNA libraries for the Ago2-immunoprecipitated RNA were produced, and the sequences of 96 clones were analyzed. The contents of the clones are shown in Figure 1A. Although significant amount of contamination by junk cDNA fragments derived from adaptor or primer sequences has been reported in other cloning methods [24], no similar contamination was found in the library made with our method. Moreover, marked reduction in the number of rRNA and tRNA clones was observed, compared to the other systems [24]. The profiles of cloned miRNA are listed in Figure 1B. Though the number of clones examined here may still be insufficient, these results suggest the usefulness of our Ago2-immunoprecipitation/cloning system of Ago2-RISC-associated miRNAs.


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

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

Categories of cDNA clones derived from immunoprecipitated small RNA of HeLa cells. A: Composition of small RNA cDNA clones. The composition of cDNA clones is shown in a circle graph. Almost all of the recovered cDNA was miRNA (94%), while the remainder was rRNA and tRNA clones (3% and 2%). 'Others' indicates the cDNA clones with hits in the genomic sequence. B: Contents of miRNA clones. Each miRNA clone was deduced by BLAST search. The y-axis indicates number of clones of each miRNA species.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Categories of cDNA clones derived from immunoprecipitated small RNA of HeLa cells. A: Composition of small RNA cDNA clones. The composition of cDNA clones is shown in a circle graph. Almost all of the recovered cDNA was miRNA (94%), while the remainder was rRNA and tRNA clones (3% and 2%). 'Others' indicates the cDNA clones with hits in the genomic sequence. B: Contents of miRNA clones. Each miRNA clone was deduced by BLAST search. The y-axis indicates number of clones of each miRNA species.
Mentions: The cDNA of the isolated small RNA was synthesized by improved adaptor ligation protocol see Methods section. We utilized the unique features of ThermoPhage™ single-stranded DNA ligase, which has ligase activities for both single-stranded RNA and DNA and can be used at 60°C [23]; this ligase was used for all of the adaptor ligation. High temperature may be advantageous for the ligation of nucleotides with tight tertiary structures. Using this method, cDNA libraries for the Ago2-immunoprecipitated RNA were produced, and the sequences of 96 clones were analyzed. The contents of the clones are shown in Figure 1A. Although significant amount of contamination by junk cDNA fragments derived from adaptor or primer sequences has been reported in other cloning methods [24], no similar contamination was found in the library made with our method. Moreover, marked reduction in the number of rRNA and tRNA clones was observed, compared to the other systems [24]. The profiles of cloned miRNA are listed in Figure 1B. Though the number of clones examined here may still be insufficient, these results suggest the usefulness of our Ago2-immunoprecipitation/cloning system of Ago2-RISC-associated miRNAs.

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.