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RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat.

Yao Y, Jin S, Long H, Yu Y, Zhang Z, Cheng G, Xu C, Ding Y, Guan Q, Li N, Fu S, Chen XJ, Yan YB, Zhang H, Tong P, Tan Y, Yu Y, Fu S, Li J, He GJ, Wu Q - Nucleic Acids Res. (2015)

Bottom Line: This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe.Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect.One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China.

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The pattern of the pairing segment of RNAe determines its enhancement efficiency of target protein expression. (A) Sketch map and detailed pairing pattern of original RNAe (the same pairing rule with AS Uchl1). (B and C) Importance of 5' TR or 5' UTR in pairing segments. Structure of pairing segments having only 5' TR or 5' UTR (B) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (C). (D and E) Length effect of 5' TR on RNAe enhancement effect in HEK293T cells. Structure of extended 5' TR pairing segments (D) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (E). (F and G) Optimization of the pairing segment of RNAe. Structure of optimized pairing segments (F) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (G). The horizontal axis indicates numbers of nucleotides pairing to 5' UTR and 5' TR of target mRNA, respectively (mean ± SD, n = 4. *P < 0.05, **P < 0.01, two tailed t-test).
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Figure 5: The pattern of the pairing segment of RNAe determines its enhancement efficiency of target protein expression. (A) Sketch map and detailed pairing pattern of original RNAe (the same pairing rule with AS Uchl1). (B and C) Importance of 5' TR or 5' UTR in pairing segments. Structure of pairing segments having only 5' TR or 5' UTR (B) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (C). (D and E) Length effect of 5' TR on RNAe enhancement effect in HEK293T cells. Structure of extended 5' TR pairing segments (D) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (E). (F and G) Optimization of the pairing segment of RNAe. Structure of optimized pairing segments (F) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (G). The horizontal axis indicates numbers of nucleotides pairing to 5' UTR and 5' TR of target mRNA, respectively (mean ± SD, n = 4. *P < 0.05, **P < 0.01, two tailed t-test).

Mentions: As the original RNAe pairs with the −40 nt to +32 nt region of the target mRNA (Figure 5A), we used the RNAe-egfpc1 with the same pairing segment length (expressed as 40:32) as the original control and a 0:0 pairing sequence (no pairing) as mock control. We further tested several RNAe constructs with pairing complementary only for the 5' untranslated region (5' UTR, 40:0 and 100:0), only the 5'-translated region (5' TR, 0:32, 0:100 and 0:200) (Figure 5B), or for the extended 5' TR (40:360, 40:560 and 40:760) (Figure 5D). These series of RNAe plasmids were co-transfected with pEGFP-C1 into HEK293T cells and EGFP green fluorescence was measured as already described. The results clearly show that the pairing sequence in the 5' TR is essential, as removing it (40:0) abolished the RNAe effect. Interestingly, prolonging the pairing in the 5' UTR (100:0) did not help; in contrast, a deletion of the pairing sequence in the 5' UTR had little influence on RNAe as long as the total length amounted to a threshold value such as 100 (0:100 or 0:200) (Figure 5C). Adding longer pairing sequences at 5' TR up to a certain length (total 400 nt) produced similar enhancement effect as the original 40:32. However, extending the pairing sequence up to 600 nt or 800 nt total produced only moderate or even inhibitory effects on target protein expression (Figure 5E). This might be due to the typical antisense pairing induced mRNA degradation mechanism. These results indicate that the original 40:32 pairing sequence is sufficient in terms of its length, region coverage and enhancement effect. We nevertheless do believe that optimization for a different protein may yield a better enhancement effect with a pairing distribution somewhat different from this standard 40:32 (Figure 5F and G). Since it was shown to be sufficient and robust, the 72 nt pairing sequence in 40:32 distribution was kept as the standard pairing sequence architecture for other RNAe experiments.


RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat.

Yao Y, Jin S, Long H, Yu Y, Zhang Z, Cheng G, Xu C, Ding Y, Guan Q, Li N, Fu S, Chen XJ, Yan YB, Zhang H, Tong P, Tan Y, Yu Y, Fu S, Li J, He GJ, Wu Q - Nucleic Acids Res. (2015)

The pattern of the pairing segment of RNAe determines its enhancement efficiency of target protein expression. (A) Sketch map and detailed pairing pattern of original RNAe (the same pairing rule with AS Uchl1). (B and C) Importance of 5' TR or 5' UTR in pairing segments. Structure of pairing segments having only 5' TR or 5' UTR (B) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (C). (D and E) Length effect of 5' TR on RNAe enhancement effect in HEK293T cells. Structure of extended 5' TR pairing segments (D) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (E). (F and G) Optimization of the pairing segment of RNAe. Structure of optimized pairing segments (F) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (G). The horizontal axis indicates numbers of nucleotides pairing to 5' UTR and 5' TR of target mRNA, respectively (mean ± SD, n = 4. *P < 0.05, **P < 0.01, two tailed t-test).
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Related In: Results  -  Collection

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Figure 5: The pattern of the pairing segment of RNAe determines its enhancement efficiency of target protein expression. (A) Sketch map and detailed pairing pattern of original RNAe (the same pairing rule with AS Uchl1). (B and C) Importance of 5' TR or 5' UTR in pairing segments. Structure of pairing segments having only 5' TR or 5' UTR (B) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (C). (D and E) Length effect of 5' TR on RNAe enhancement effect in HEK293T cells. Structure of extended 5' TR pairing segments (D) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (E). (F and G) Optimization of the pairing segment of RNAe. Structure of optimized pairing segments (F) and their effect on EGFP expression indicated by EGFP fluorescence in HEK293T cells (G). The horizontal axis indicates numbers of nucleotides pairing to 5' UTR and 5' TR of target mRNA, respectively (mean ± SD, n = 4. *P < 0.05, **P < 0.01, two tailed t-test).
Mentions: As the original RNAe pairs with the −40 nt to +32 nt region of the target mRNA (Figure 5A), we used the RNAe-egfpc1 with the same pairing segment length (expressed as 40:32) as the original control and a 0:0 pairing sequence (no pairing) as mock control. We further tested several RNAe constructs with pairing complementary only for the 5' untranslated region (5' UTR, 40:0 and 100:0), only the 5'-translated region (5' TR, 0:32, 0:100 and 0:200) (Figure 5B), or for the extended 5' TR (40:360, 40:560 and 40:760) (Figure 5D). These series of RNAe plasmids were co-transfected with pEGFP-C1 into HEK293T cells and EGFP green fluorescence was measured as already described. The results clearly show that the pairing sequence in the 5' TR is essential, as removing it (40:0) abolished the RNAe effect. Interestingly, prolonging the pairing in the 5' UTR (100:0) did not help; in contrast, a deletion of the pairing sequence in the 5' UTR had little influence on RNAe as long as the total length amounted to a threshold value such as 100 (0:100 or 0:200) (Figure 5C). Adding longer pairing sequences at 5' TR up to a certain length (total 400 nt) produced similar enhancement effect as the original 40:32. However, extending the pairing sequence up to 600 nt or 800 nt total produced only moderate or even inhibitory effects on target protein expression (Figure 5E). This might be due to the typical antisense pairing induced mRNA degradation mechanism. These results indicate that the original 40:32 pairing sequence is sufficient in terms of its length, region coverage and enhancement effect. We nevertheless do believe that optimization for a different protein may yield a better enhancement effect with a pairing distribution somewhat different from this standard 40:32 (Figure 5F and G). Since it was shown to be sufficient and robust, the 72 nt pairing sequence in 40:32 distribution was kept as the standard pairing sequence architecture for other RNAe experiments.

Bottom Line: This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe.Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect.One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Show MeSH
Related in: MedlinePlus