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Uracils at nucleotide position 9-11 are required for the rapid turnover of miR-29 family.

Zhang Z, Zou J, Wang GK, Zhang JT, Huang S, Qin YW, Jing Q - Nucleic Acids Res. (2011)

Bottom Line: Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence.The effect of uracil-rich sequence on microRNA turnover depends on the sequence context.The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Changhai Hospital, Shanghai, China.

ABSTRACT
MicroRNAs are endogenous small RNA molecules that regulate gene expression. Although the biogenesis of microRNAs and their regulation have been thoroughly elucidated, the degradation of microRNAs has not been fully understood. Here by using the pulse-chase approach, we performed the direct measurement of microRNA lifespan. Five representative microRNAs demonstrated a general feature of relatively long lifespan. However, the decay dynamic varies considerably between these individual microRNAs. Mutation analysis of miR-29b sequence revealed that uracils at nucleotide position 9-11 are required for its rapid decay, in that both specific nucleotides and their position are critical. The effect of uracil-rich element on miR-29b decay dynamic occurs in duplex but not in single strand RNA. Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence. Among them, Northern blotting shows that miR-29c and fruit fly bantam possess a relatively rapid turnover rate. The effect of uracil-rich sequence on microRNA turnover depends on the sequence context. The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.

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Synthetic double-strand RNAs as miRNA mimickers for studying the stability of mature miRNAs. (A) 40 nM synthetic miRNA mimickers were transfected into HeLa cells for 4 h and the cellular abundance of each miRNA was assayed by real-time PCR. Relative abundance shows the relative enrichment fold of miRNA quantity after transfection of miRNA mimickers to with a negative control RNA (NC). (B) miRNA mimickers for miR-29 family (miR-29a/b/c) were able to suppress the luciferase reporter with 3′-UTR of its target mRNA cdc42 in HeLa cells. (C) Left: miR-29b mimickers with indicated concentration were transfected into HeLa cells and quantified by either northern blotting (left) or qPCR (right). Cellular abundance of miRNA is proportionally to the transfection dose at the range indicated. Luciferase assay and PCR was repeated independently three times, and northern blotting two times.
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Figure 1: Synthetic double-strand RNAs as miRNA mimickers for studying the stability of mature miRNAs. (A) 40 nM synthetic miRNA mimickers were transfected into HeLa cells for 4 h and the cellular abundance of each miRNA was assayed by real-time PCR. Relative abundance shows the relative enrichment fold of miRNA quantity after transfection of miRNA mimickers to with a negative control RNA (NC). (B) miRNA mimickers for miR-29 family (miR-29a/b/c) were able to suppress the luciferase reporter with 3′-UTR of its target mRNA cdc42 in HeLa cells. (C) Left: miR-29b mimickers with indicated concentration were transfected into HeLa cells and quantified by either northern blotting (left) or qPCR (right). Cellular abundance of miRNA is proportionally to the transfection dose at the range indicated. Luciferase assay and PCR was repeated independently three times, and northern blotting two times.

Mentions: Both transcription and post-transcriptional processing influence miRNAs generation, and hence transcriptional shutoff may not completely terminate the generation of miRNAs. We therefore chose to employ the pulse–chase assay with synthetic RNA duplex as miRNA mimickers to examine the miRNA turnover in cell culture (24). Synthetic duplex of miRNA mimickers in our assay are completely complementary. First, we examined whether these miRNA mimickers could steadily accumulate in cells and therefore minimize the interference by endogenous miRNA species. Five microRNAs with different endogenous expression level were selected (25), and their mimickers were transfected into HeLa and analyzed by real-time PCR. A synthetic duplex RNA, which does not target any RNAs, served as the negative control (NC). Each miRNA increased substantially and therefore allow a long-term chase (Figure 1A). Previously it has been proven that this type of synthetic miRNA mimickers could incorporate into the RNA-induced silencing complex (RISC) (13). Here to further validate whether these mimickers could simulate miRNAs in a cellular context, we examined the silencing effect of synthetic miRNA mimickers on target mRNAs. MiR-29 family, including miR-29a, b and c, was reported to directly target cdc42 mRNA in HeLa cells (26). Our reporter assay showed that the synthetic miR-29 mixture (equal amount of miR-29a/b/c) was able to repress the luciferase reporter fused to the cdc42 3′-UTR by 40% (Figure 1B). A miR-29 mutant with the substitution of uracil at nucleotide position 10 with cytosine (miR-29b-C10) was as effective as the wild-type.Figure 1.


Uracils at nucleotide position 9-11 are required for the rapid turnover of miR-29 family.

Zhang Z, Zou J, Wang GK, Zhang JT, Huang S, Qin YW, Jing Q - Nucleic Acids Res. (2011)

Synthetic double-strand RNAs as miRNA mimickers for studying the stability of mature miRNAs. (A) 40 nM synthetic miRNA mimickers were transfected into HeLa cells for 4 h and the cellular abundance of each miRNA was assayed by real-time PCR. Relative abundance shows the relative enrichment fold of miRNA quantity after transfection of miRNA mimickers to with a negative control RNA (NC). (B) miRNA mimickers for miR-29 family (miR-29a/b/c) were able to suppress the luciferase reporter with 3′-UTR of its target mRNA cdc42 in HeLa cells. (C) Left: miR-29b mimickers with indicated concentration were transfected into HeLa cells and quantified by either northern blotting (left) or qPCR (right). Cellular abundance of miRNA is proportionally to the transfection dose at the range indicated. Luciferase assay and PCR was repeated independently three times, and northern blotting two times.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105410&req=5

Figure 1: Synthetic double-strand RNAs as miRNA mimickers for studying the stability of mature miRNAs. (A) 40 nM synthetic miRNA mimickers were transfected into HeLa cells for 4 h and the cellular abundance of each miRNA was assayed by real-time PCR. Relative abundance shows the relative enrichment fold of miRNA quantity after transfection of miRNA mimickers to with a negative control RNA (NC). (B) miRNA mimickers for miR-29 family (miR-29a/b/c) were able to suppress the luciferase reporter with 3′-UTR of its target mRNA cdc42 in HeLa cells. (C) Left: miR-29b mimickers with indicated concentration were transfected into HeLa cells and quantified by either northern blotting (left) or qPCR (right). Cellular abundance of miRNA is proportionally to the transfection dose at the range indicated. Luciferase assay and PCR was repeated independently three times, and northern blotting two times.
Mentions: Both transcription and post-transcriptional processing influence miRNAs generation, and hence transcriptional shutoff may not completely terminate the generation of miRNAs. We therefore chose to employ the pulse–chase assay with synthetic RNA duplex as miRNA mimickers to examine the miRNA turnover in cell culture (24). Synthetic duplex of miRNA mimickers in our assay are completely complementary. First, we examined whether these miRNA mimickers could steadily accumulate in cells and therefore minimize the interference by endogenous miRNA species. Five microRNAs with different endogenous expression level were selected (25), and their mimickers were transfected into HeLa and analyzed by real-time PCR. A synthetic duplex RNA, which does not target any RNAs, served as the negative control (NC). Each miRNA increased substantially and therefore allow a long-term chase (Figure 1A). Previously it has been proven that this type of synthetic miRNA mimickers could incorporate into the RNA-induced silencing complex (RISC) (13). Here to further validate whether these mimickers could simulate miRNAs in a cellular context, we examined the silencing effect of synthetic miRNA mimickers on target mRNAs. MiR-29 family, including miR-29a, b and c, was reported to directly target cdc42 mRNA in HeLa cells (26). Our reporter assay showed that the synthetic miR-29 mixture (equal amount of miR-29a/b/c) was able to repress the luciferase reporter fused to the cdc42 3′-UTR by 40% (Figure 1B). A miR-29 mutant with the substitution of uracil at nucleotide position 10 with cytosine (miR-29b-C10) was as effective as the wild-type.Figure 1.

Bottom Line: Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence.The effect of uracil-rich sequence on microRNA turnover depends on the sequence context.The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Changhai Hospital, Shanghai, China.

ABSTRACT
MicroRNAs are endogenous small RNA molecules that regulate gene expression. Although the biogenesis of microRNAs and their regulation have been thoroughly elucidated, the degradation of microRNAs has not been fully understood. Here by using the pulse-chase approach, we performed the direct measurement of microRNA lifespan. Five representative microRNAs demonstrated a general feature of relatively long lifespan. However, the decay dynamic varies considerably between these individual microRNAs. Mutation analysis of miR-29b sequence revealed that uracils at nucleotide position 9-11 are required for its rapid decay, in that both specific nucleotides and their position are critical. The effect of uracil-rich element on miR-29b decay dynamic occurs in duplex but not in single strand RNA. Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence. Among them, Northern blotting shows that miR-29c and fruit fly bantam possess a relatively rapid turnover rate. The effect of uracil-rich sequence on microRNA turnover depends on the sequence context. The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.

Show MeSH
Related in: MedlinePlus