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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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Related in: MedlinePlus

Uracils at nucleotides from 9 to 11 are required for the rapid decay of miR-29b. (A) Sequence of miR-29a/b and mutants. Nucleotides under investigation are underlined and uracils are marked as lowercase. (B) Northern blotting of indicated miRNAs in pulse–chase assay. The northern blot image was showed in the left panel and the quantification of bands in northern blotting images was displayed in the right panel. Intensity of miRNAs was normalized to U6. MiR-29a is much stable than miR-29b. Substitution of uracil at position 10 with cytosine for miR-29b (miR-29b-C10) has a detectable effect on its decay rate, while substitutions of nucleotides at both 9–10, 10–11 and from 9 to 11 (miR-29b-C9C10, -C10C11, -C9C10C11) significantly enhance the miRNA stability. Substitution with adenosine (miR-29b-A9A10A11) also delayed the decay. Movement of uracils from positions 9 to 11 to most near position (miR-29b-U12U13U14) stabilized the miRNA. (C) Upper: miR-29b mimicker (duplex) was transfected into HeLa cells for 4 h and the star strand of the mimicker was detected at the indicated time points. The star strand could not be detected unless film was over-exposed. Lower: Single strand of miR-29b and miR-29b mutant (miR-29b-C9C10C11) was transfected and chased by northern blotting. Both were at a rapid decay rate. Three independent experiments for each miRNA were performed and the typical one was showed here.
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Figure 3: Uracils at nucleotides from 9 to 11 are required for the rapid decay of miR-29b. (A) Sequence of miR-29a/b and mutants. Nucleotides under investigation are underlined and uracils are marked as lowercase. (B) Northern blotting of indicated miRNAs in pulse–chase assay. The northern blot image was showed in the left panel and the quantification of bands in northern blotting images was displayed in the right panel. Intensity of miRNAs was normalized to U6. MiR-29a is much stable than miR-29b. Substitution of uracil at position 10 with cytosine for miR-29b (miR-29b-C10) has a detectable effect on its decay rate, while substitutions of nucleotides at both 9–10, 10–11 and from 9 to 11 (miR-29b-C9C10, -C10C11, -C9C10C11) significantly enhance the miRNA stability. Substitution with adenosine (miR-29b-A9A10A11) also delayed the decay. Movement of uracils from positions 9 to 11 to most near position (miR-29b-U12U13U14) stabilized the miRNA. (C) Upper: miR-29b mimicker (duplex) was transfected into HeLa cells for 4 h and the star strand of the mimicker was detected at the indicated time points. The star strand could not be detected unless film was over-exposed. Lower: Single strand of miR-29b and miR-29b mutant (miR-29b-C9C10C11) was transfected and chased by northern blotting. Both were at a rapid decay rate. Three independent experiments for each miRNA were performed and the typical one was showed here.

Mentions: MiR-29a is stable, while miR-29b is rapidly decayed in HeLa cells although their sequence are highly similar (13), thus we took miR-29 family as a paradigm to study the sequence dependence of miRNA degradation. MiR-29a and miR-29b differ at two regions. One is at their 3′-end and the other is at nucleotide position 10, with cytosine in miR-29a and uracil in miR-29b (Figure 3A). It has already been proven that the sequence at miR-29b 3′-end alone is not responsible for its rapid decay (13). We asked whether the variance at position 10 together with near nucleotides is involved in miRNA inherent stability. At the beginning, uracil was mutated into cytosine for miR-29b (miR-29b-C10), and this mutation did slightly enhance miRNA stability (Figure 3B). We further questioned whether uracils near position 10 had a synergic effect with the central uracil (U10) to function. Mutation of uracils in both position 9 and 10 (miR-29b-C9C10) into cytosine prolonged the half life of miR-29b (Figure 3B). This effect was observed for mutation in both position 10 and 11 (miR-29b-C10C11) to a more significant degree. All three uracils from position 9 to 11 into cytosine (miR-29b-C9C10C11) abolished the rapid decay of miR-29b (Figure 3B). It is questionable whether the change of observed decay rate is simply attributed to the alteration of miRNA GC content. Mutation into adenosine (miR-29b-A9A10A11) was examined and the result demonstrated a significant decrease in the decay rate (Figure 3B). Furthermore, to test whether position at nucleotides 9–11 is critical for the effect of uracil stretch, the nucleotides at position 9–11 were exchanged with that at position 12–14 (miR-29b-U12U13U14, see Figure 3A). Northern blotting showed that the change in position stabilized the wild-type miR-29b (Figure 3B). To test whether our observation would hold generally, the lifespan of miR-29b and miR-29b-C9C10C11 was chased in HEK293T cells. Indeed, mutation of centric uracils impeded the rapid degradation of miR-29b (Supplementary Figure S2). Our observation therefore demonstrated that uracil at the middle region of miR-29b (from 9 to 11 nt) was required for its rapid turnover.Figure 3.


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)

Uracils at nucleotides from 9 to 11 are required for the rapid decay of miR-29b. (A) Sequence of miR-29a/b and mutants. Nucleotides under investigation are underlined and uracils are marked as lowercase. (B) Northern blotting of indicated miRNAs in pulse–chase assay. The northern blot image was showed in the left panel and the quantification of bands in northern blotting images was displayed in the right panel. Intensity of miRNAs was normalized to U6. MiR-29a is much stable than miR-29b. Substitution of uracil at position 10 with cytosine for miR-29b (miR-29b-C10) has a detectable effect on its decay rate, while substitutions of nucleotides at both 9–10, 10–11 and from 9 to 11 (miR-29b-C9C10, -C10C11, -C9C10C11) significantly enhance the miRNA stability. Substitution with adenosine (miR-29b-A9A10A11) also delayed the decay. Movement of uracils from positions 9 to 11 to most near position (miR-29b-U12U13U14) stabilized the miRNA. (C) Upper: miR-29b mimicker (duplex) was transfected into HeLa cells for 4 h and the star strand of the mimicker was detected at the indicated time points. The star strand could not be detected unless film was over-exposed. Lower: Single strand of miR-29b and miR-29b mutant (miR-29b-C9C10C11) was transfected and chased by northern blotting. Both were at a rapid decay rate. Three independent experiments for each miRNA were performed and the typical one was showed here.
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Figure 3: Uracils at nucleotides from 9 to 11 are required for the rapid decay of miR-29b. (A) Sequence of miR-29a/b and mutants. Nucleotides under investigation are underlined and uracils are marked as lowercase. (B) Northern blotting of indicated miRNAs in pulse–chase assay. The northern blot image was showed in the left panel and the quantification of bands in northern blotting images was displayed in the right panel. Intensity of miRNAs was normalized to U6. MiR-29a is much stable than miR-29b. Substitution of uracil at position 10 with cytosine for miR-29b (miR-29b-C10) has a detectable effect on its decay rate, while substitutions of nucleotides at both 9–10, 10–11 and from 9 to 11 (miR-29b-C9C10, -C10C11, -C9C10C11) significantly enhance the miRNA stability. Substitution with adenosine (miR-29b-A9A10A11) also delayed the decay. Movement of uracils from positions 9 to 11 to most near position (miR-29b-U12U13U14) stabilized the miRNA. (C) Upper: miR-29b mimicker (duplex) was transfected into HeLa cells for 4 h and the star strand of the mimicker was detected at the indicated time points. The star strand could not be detected unless film was over-exposed. Lower: Single strand of miR-29b and miR-29b mutant (miR-29b-C9C10C11) was transfected and chased by northern blotting. Both were at a rapid decay rate. Three independent experiments for each miRNA were performed and the typical one was showed here.
Mentions: MiR-29a is stable, while miR-29b is rapidly decayed in HeLa cells although their sequence are highly similar (13), thus we took miR-29 family as a paradigm to study the sequence dependence of miRNA degradation. MiR-29a and miR-29b differ at two regions. One is at their 3′-end and the other is at nucleotide position 10, with cytosine in miR-29a and uracil in miR-29b (Figure 3A). It has already been proven that the sequence at miR-29b 3′-end alone is not responsible for its rapid decay (13). We asked whether the variance at position 10 together with near nucleotides is involved in miRNA inherent stability. At the beginning, uracil was mutated into cytosine for miR-29b (miR-29b-C10), and this mutation did slightly enhance miRNA stability (Figure 3B). We further questioned whether uracils near position 10 had a synergic effect with the central uracil (U10) to function. Mutation of uracils in both position 9 and 10 (miR-29b-C9C10) into cytosine prolonged the half life of miR-29b (Figure 3B). This effect was observed for mutation in both position 10 and 11 (miR-29b-C10C11) to a more significant degree. All three uracils from position 9 to 11 into cytosine (miR-29b-C9C10C11) abolished the rapid decay of miR-29b (Figure 3B). It is questionable whether the change of observed decay rate is simply attributed to the alteration of miRNA GC content. Mutation into adenosine (miR-29b-A9A10A11) was examined and the result demonstrated a significant decrease in the decay rate (Figure 3B). Furthermore, to test whether position at nucleotides 9–11 is critical for the effect of uracil stretch, the nucleotides at position 9–11 were exchanged with that at position 12–14 (miR-29b-U12U13U14, see Figure 3A). Northern blotting showed that the change in position stabilized the wild-type miR-29b (Figure 3B). To test whether our observation would hold generally, the lifespan of miR-29b and miR-29b-C9C10C11 was chased in HEK293T cells. Indeed, mutation of centric uracils impeded the rapid degradation of miR-29b (Supplementary Figure S2). Our observation therefore demonstrated that uracil at the middle region of miR-29b (from 9 to 11 nt) was required for its rapid turnover.Figure 3.

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