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Decreased dicer expression enhances SRP-mediated protein targeting.

Ren YF, Li G, Xue YF, Zhang XJ, Song YJ, Lv L, Wu J, Fang YX, Wang YQ, Shi KQ, Chen YP, Tang KF - PLoS ONE (2013)

Bottom Line: Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation.These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting.Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genomic Medicine, Wenzhou Medical College, Wenzhou, Zhejiang Province, PR China.

ABSTRACT
We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

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The 7SL RNA fragment mixture partially restores SRP function in Dicer-knockdown cells.(A) HepG2.2.15 cells were co-transfected for 48 h with the pSEAP2-control and either shDCR or shCon plasmids. The Dicer-knockdown cells (K/D) were then transfected with the 7SL RNA fragment mixture (7SL mix) or LacZ RNA, and SEAP activity was determined 48 h after the second transfection. *p<0.05, **p<0.01. (B) HEK293T cells that stably expressed ECFP-ER were pretreated with siDCR for 48 h, and then co-transfected with 7SL mix or LacZ RNA and siDCR. Fluorescence was detected 48 h after the second transfection. Cells treated twice with siDCR (K/D) or siCon (Con) served as controls. (C) HepG2.2.15 cells pretreated with siDCR for 48 h were co-transfected with 7SL mix and siDCR. The levels of cell surface glycoproteins were measured 48 h after the second transfection. Cells treated twice with siDCR or siCon served as controls. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the red line represents cells transfected with siCon, the black line represents cells transfected with siDCR, and the blue line represents cells co-transfected with siDCR and the 7SL mix.
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pone-0056950-g004: The 7SL RNA fragment mixture partially restores SRP function in Dicer-knockdown cells.(A) HepG2.2.15 cells were co-transfected for 48 h with the pSEAP2-control and either shDCR or shCon plasmids. The Dicer-knockdown cells (K/D) were then transfected with the 7SL RNA fragment mixture (7SL mix) or LacZ RNA, and SEAP activity was determined 48 h after the second transfection. *p<0.05, **p<0.01. (B) HEK293T cells that stably expressed ECFP-ER were pretreated with siDCR for 48 h, and then co-transfected with 7SL mix or LacZ RNA and siDCR. Fluorescence was detected 48 h after the second transfection. Cells treated twice with siDCR (K/D) or siCon (Con) served as controls. (C) HepG2.2.15 cells pretreated with siDCR for 48 h were co-transfected with 7SL mix and siDCR. The levels of cell surface glycoproteins were measured 48 h after the second transfection. Cells treated twice with siDCR or siCon served as controls. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the red line represents cells transfected with siCon, the black line represents cells transfected with siDCR, and the blue line represents cells co-transfected with siDCR and the 7SL mix.

Mentions: To further investigate whether enhanced SRP function in Dicer knockdown cells is due to reduction of Dicer-processed 7SL RNAs, we transfected Dicer-knockdown cells with a mixture of the 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299). The 7SL RNA fragment mixture not only partially inhibited the secretion of SEAP (Fig. 4A), but also partially restored the levels of ECFP-ER and cell surface glycoproteins in Dicer-knockdown cells (Fig. 4B and 4C). In contrast, transfection of Dicer-knockdown cells with LacZ RNA had no detectable effect (Fig. 4A, 4B, and data not shown).


Decreased dicer expression enhances SRP-mediated protein targeting.

Ren YF, Li G, Xue YF, Zhang XJ, Song YJ, Lv L, Wu J, Fang YX, Wang YQ, Shi KQ, Chen YP, Tang KF - PLoS ONE (2013)

The 7SL RNA fragment mixture partially restores SRP function in Dicer-knockdown cells.(A) HepG2.2.15 cells were co-transfected for 48 h with the pSEAP2-control and either shDCR or shCon plasmids. The Dicer-knockdown cells (K/D) were then transfected with the 7SL RNA fragment mixture (7SL mix) or LacZ RNA, and SEAP activity was determined 48 h after the second transfection. *p<0.05, **p<0.01. (B) HEK293T cells that stably expressed ECFP-ER were pretreated with siDCR for 48 h, and then co-transfected with 7SL mix or LacZ RNA and siDCR. Fluorescence was detected 48 h after the second transfection. Cells treated twice with siDCR (K/D) or siCon (Con) served as controls. (C) HepG2.2.15 cells pretreated with siDCR for 48 h were co-transfected with 7SL mix and siDCR. The levels of cell surface glycoproteins were measured 48 h after the second transfection. Cells treated twice with siDCR or siCon served as controls. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the red line represents cells transfected with siCon, the black line represents cells transfected with siDCR, and the blue line represents cells co-transfected with siDCR and the 7SL mix.
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Related In: Results  -  Collection

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pone-0056950-g004: The 7SL RNA fragment mixture partially restores SRP function in Dicer-knockdown cells.(A) HepG2.2.15 cells were co-transfected for 48 h with the pSEAP2-control and either shDCR or shCon plasmids. The Dicer-knockdown cells (K/D) were then transfected with the 7SL RNA fragment mixture (7SL mix) or LacZ RNA, and SEAP activity was determined 48 h after the second transfection. *p<0.05, **p<0.01. (B) HEK293T cells that stably expressed ECFP-ER were pretreated with siDCR for 48 h, and then co-transfected with 7SL mix or LacZ RNA and siDCR. Fluorescence was detected 48 h after the second transfection. Cells treated twice with siDCR (K/D) or siCon (Con) served as controls. (C) HepG2.2.15 cells pretreated with siDCR for 48 h were co-transfected with 7SL mix and siDCR. The levels of cell surface glycoproteins were measured 48 h after the second transfection. Cells treated twice with siDCR or siCon served as controls. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the red line represents cells transfected with siCon, the black line represents cells transfected with siDCR, and the blue line represents cells co-transfected with siDCR and the 7SL mix.
Mentions: To further investigate whether enhanced SRP function in Dicer knockdown cells is due to reduction of Dicer-processed 7SL RNAs, we transfected Dicer-knockdown cells with a mixture of the 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299). The 7SL RNA fragment mixture not only partially inhibited the secretion of SEAP (Fig. 4A), but also partially restored the levels of ECFP-ER and cell surface glycoproteins in Dicer-knockdown cells (Fig. 4B and 4C). In contrast, transfection of Dicer-knockdown cells with LacZ RNA had no detectable effect (Fig. 4A, 4B, and data not shown).

Bottom Line: Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation.These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting.Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genomic Medicine, Wenzhou Medical College, Wenzhou, Zhejiang Province, PR China.

ABSTRACT
We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

Show MeSH
Related in: MedlinePlus