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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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7SL RNA fragments repress SRP-mediated protein targeting.(A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different 7SL RNA fragments or the control RNA (LacZ RNA), and SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 compared with control RNA. (B) HEK293T cells stably expressing ECFP-ER were transfected with different 7SL RNA fragments or LacZ RNA. Fluorescence was detected 48 h after transfection. (C) HepG2.2.15 cells were transfected with different 7SL RNA fragments or LacZ RNA, and cell surface glycoproteins were measured 48 h after transfection. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents cells transfected with LacZ RNA, and the dashed line represents cells transfected with the 7SL RNA fragment.
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pone-0056950-g001: 7SL RNA fragments repress SRP-mediated protein targeting.(A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different 7SL RNA fragments or the control RNA (LacZ RNA), and SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 compared with control RNA. (B) HEK293T cells stably expressing ECFP-ER were transfected with different 7SL RNA fragments or LacZ RNA. Fluorescence was detected 48 h after transfection. (C) HepG2.2.15 cells were transfected with different 7SL RNA fragments or LacZ RNA, and cell surface glycoproteins were measured 48 h after transfection. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents cells transfected with LacZ RNA, and the dashed line represents cells transfected with the 7SL RNA fragment.

Mentions: We reported that in addition to the ∼20 nt small RNAs (including 7SL sRNA5cd and 7SL sRNA8b), 7SL RNA is also processed by Dicer into longer fragments [16]. We developed a method to clone these long 7SL RNA fragments (Fig. S2). Sequence analysis of eight clones revealed that two contain an inserted sequence corresponding to nucleotides 1 to 96 of 7SL RNA. This result is consistent with our previous findings [16], which indicate that Dicer cleaves 7SL RNA at four major sites (Fig. S3A). To address whether these long Dicer-processed 7SL fragments can modulate SRP-mediated protein targeting, we synthesized three 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299) (Fig. S3B). Co-transfection experiment indicated that 7SL(1-96) inhibited SEAP secretion in a concentration-dependent manner (Fig. S4). Transfection of 7SL (1-96), 7SL(1-212) or 7SL(97-299) (80 nM each) significantly inhibited the secretion of SEAP (Figs. 1A and S5) as well as the expression of ECFP-ER and cell surface glycoproteins (Figs. 1B and 1C).


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)

7SL RNA fragments repress SRP-mediated protein targeting.(A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different 7SL RNA fragments or the control RNA (LacZ RNA), and SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 compared with control RNA. (B) HEK293T cells stably expressing ECFP-ER were transfected with different 7SL RNA fragments or LacZ RNA. Fluorescence was detected 48 h after transfection. (C) HepG2.2.15 cells were transfected with different 7SL RNA fragments or LacZ RNA, and cell surface glycoproteins were measured 48 h after transfection. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents cells transfected with LacZ RNA, and the dashed line represents cells transfected with the 7SL RNA fragment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585229&req=5

pone-0056950-g001: 7SL RNA fragments repress SRP-mediated protein targeting.(A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different 7SL RNA fragments or the control RNA (LacZ RNA), and SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 compared with control RNA. (B) HEK293T cells stably expressing ECFP-ER were transfected with different 7SL RNA fragments or LacZ RNA. Fluorescence was detected 48 h after transfection. (C) HepG2.2.15 cells were transfected with different 7SL RNA fragments or LacZ RNA, and cell surface glycoproteins were measured 48 h after transfection. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents cells transfected with LacZ RNA, and the dashed line represents cells transfected with the 7SL RNA fragment.
Mentions: We reported that in addition to the ∼20 nt small RNAs (including 7SL sRNA5cd and 7SL sRNA8b), 7SL RNA is also processed by Dicer into longer fragments [16]. We developed a method to clone these long 7SL RNA fragments (Fig. S2). Sequence analysis of eight clones revealed that two contain an inserted sequence corresponding to nucleotides 1 to 96 of 7SL RNA. This result is consistent with our previous findings [16], which indicate that Dicer cleaves 7SL RNA at four major sites (Fig. S3A). To address whether these long Dicer-processed 7SL fragments can modulate SRP-mediated protein targeting, we synthesized three 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299) (Fig. S3B). Co-transfection experiment indicated that 7SL(1-96) inhibited SEAP secretion in a concentration-dependent manner (Fig. S4). Transfection of 7SL (1-96), 7SL(1-212) or 7SL(97-299) (80 nM each) significantly inhibited the secretion of SEAP (Figs. 1A and S5) as well as the expression of ECFP-ER and cell surface glycoproteins (Figs. 1B and 1C).

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