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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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Dicer-knockdown enhances SRP-mediated protein targeting.(A) HepG2.2.15 and HEK293T cells were co-transfected with the SEAP-control and shDCR or shCon plasmids, and SEAP activity was determined 96 h later, **p<0.01 compared with the control. (B) HEK293T cells stably expressing with ECFR-ER were transfected twice with either siDCR or siCon. Fluorescence was detected 96 h after transfection. Con: control cells, K/D: Dicer knockdown cells. (C) HepG2.2.15 and HEK293T cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer-knockdown cells, and dashed line represents the control cells.
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pone-0056950-g003: Dicer-knockdown enhances SRP-mediated protein targeting.(A) HepG2.2.15 and HEK293T cells were co-transfected with the SEAP-control and shDCR or shCon plasmids, and SEAP activity was determined 96 h later, **p<0.01 compared with the control. (B) HEK293T cells stably expressing with ECFR-ER were transfected twice with either siDCR or siCon. Fluorescence was detected 96 h after transfection. Con: control cells, K/D: Dicer knockdown cells. (C) HepG2.2.15 and HEK293T cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer-knockdown cells, and dashed line represents the control cells.

Mentions: We reported that Dicer-processed 7SL RNA fragments are reduced in Dicer-knockdown cells [16]. Here we show that some 7SL RNA fragments function as dominant-negative regulators of full-length 7SL RNA and inhibit SRP-mediated protein targeting (Figs. 1 and 2). Therefore, we hypothesized that Dicer knockdown may enhance SRP-mediated protein targeting. To test this hypothesis, we knocked-down Dicer expression in HepG2.2.15 and HEK293T cells (Fig. S6). Knockdown of Dicer significantly increased the secretion of SEAP (Fig. 3A) as well as the expression of ECFP-ER and cell surface glycoproteins (Fig. 3B and 3C). Similar results were obtained using additional cell lines (Fig. S6 and S7), which indicates that the effect of Dicer on SRP-mediated protein targeting is not cell-type specific.


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)

Dicer-knockdown enhances SRP-mediated protein targeting.(A) HepG2.2.15 and HEK293T cells were co-transfected with the SEAP-control and shDCR or shCon plasmids, and SEAP activity was determined 96 h later, **p<0.01 compared with the control. (B) HEK293T cells stably expressing with ECFR-ER were transfected twice with either siDCR or siCon. Fluorescence was detected 96 h after transfection. Con: control cells, K/D: Dicer knockdown cells. (C) HepG2.2.15 and HEK293T cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer-knockdown cells, and dashed line represents the control cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585229&req=5

pone-0056950-g003: Dicer-knockdown enhances SRP-mediated protein targeting.(A) HepG2.2.15 and HEK293T cells were co-transfected with the SEAP-control and shDCR or shCon plasmids, and SEAP activity was determined 96 h later, **p<0.01 compared with the control. (B) HEK293T cells stably expressing with ECFR-ER were transfected twice with either siDCR or siCon. Fluorescence was detected 96 h after transfection. Con: control cells, K/D: Dicer knockdown cells. (C) HepG2.2.15 and HEK293T cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and the number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer-knockdown cells, and dashed line represents the control cells.
Mentions: We reported that Dicer-processed 7SL RNA fragments are reduced in Dicer-knockdown cells [16]. Here we show that some 7SL RNA fragments function as dominant-negative regulators of full-length 7SL RNA and inhibit SRP-mediated protein targeting (Figs. 1 and 2). Therefore, we hypothesized that Dicer knockdown may enhance SRP-mediated protein targeting. To test this hypothesis, we knocked-down Dicer expression in HepG2.2.15 and HEK293T cells (Fig. S6). Knockdown of Dicer significantly increased the secretion of SEAP (Fig. 3A) as well as the expression of ECFP-ER and cell surface glycoproteins (Fig. 3B and 3C). Similar results were obtained using additional cell lines (Fig. S6 and S7), which indicates that the effect of Dicer on SRP-mediated protein targeting is not cell-type specific.

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