Decreased Dicer Expression Enhances SRP-Mediated Protein Targeting

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.


Introduction
The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper subcellular compartments and is essential for cotranslational insertion of membrane and secretory proteins into the lumen of the endoplasmic reticulum (ER) [1,2]. The cotranslational protein targeting process is characterized by a well-understood sequence of events as follows: The N-terminal signal sequence of a nascent polypeptide is recognized on the ribosome by SRP, and this SRP-ribosome complex is then targeted to the membrane via the SRP receptor. Finally, the nascent chain is transferred from SRP to the protein-conducting channel, through which it is cotranslationally threaded [3].
The eukaryotic SRP complex comprises six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72) bound to an RNA molecule (the 7SL RNA) [4,5]. Down-regulation of 7SL RNA leads to inefficient targeting of nascent polypeptides to the ER. Misra and colleagues reported that in macrophage-like cell lines (J774G8 and U937), targeting of proteins to the ER and plasma membrane and the secretion of proteins are compromised by infection with Leishmania due to down-regulation of 7SL RNA [6]. Knockdown of SRP14, SRP54, or SRP72 led to reduced levels of 7SL RNA and resulted in inefficient SRP-mediated protein targeting [7].
7SL RNA was first detected in Rous sarcoma virus particles [8] and later identified as a stable component of the SRP complex [4,5]. 7SL RNA is packaged into HIV-1 virions, and an endoribonucleolytic fragment of 7SL RNA (termed 7SLrem) is present in HIV-1 virions and minimal virus-like particles [9,10,11,12,13]. The ends of 7SLrem map to bulges in the secondary structure of the full-length 7SL RNA where nucleotides remain unpaired, implying that the full-length 7SL RNA is processed into 7SLrem by a single-stranded endonuclease [10]. Recently, we found that Dicer, a double-stranded endonuclease involved in the biogenesis of microRNA (miRNA) and small interfering RNA (siRNA) [14,15], also processes 7SL RNA into fragments ranging from ,20 to more than 200 nucleotides (nt) [16]. We also showed that the ,20 nt RNAs did not function like miRNAs, nor did they regulate the expression of 7SL RNA [16].
In the present study, we investigated the cellular functions of the Dicer-processed 7SL RNA fragments and found that some function as dominant-negative regulators of the full-length 7SL RNA by interfering with the formation of the SRP complex and inhibiting SRP-mediated protein targeting.

Materials and Methods
Cell Culture, siRNAs, Plasmids and Transfection HepG2.2.15 and HEK293T cells were cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA), RKO and T29 cells were cultured in DMEM (Hyclone), and HCT116 cells were cultured in McCoy's 5A medium (Gibco, GrandIsland, NY, USA). All media were supplemented with 10% fetal bovine serum. We obtained siRNAs from Invitrogen (Life Technologies, Shanghai, China) and constructed plasmids expressing shRNAs (shDCR and shCon) by inserting the corresponding hairpin oligonucleotides into the pSUPER.neo+GFP vector (Oligoengine, Seattle, WA, USA). The target sequences of siRNAs and shRNAs were as follows: Dicer (AAGGCTTACCTTCTCCAGGCT), Control (AATTCTCCGAACGTGTCACGT). RNA transfection was performed as described [17,18,19]. The efficiency of interference was validated using either real-time RT-PCR or western blotting, or both. Plasmid transfection was performed using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions.

Western Blotting
Cells were lysed in RIPA buffer and equal amounts of denatured protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were then incubated with primary antibody, followed by horseradish peroxidase-linked secondary antibody, and immune complexes were detected using ECL plus reagent (Millipore). The primary antibodies were as follows: Dicer and SRP19 (Abcam, Cambridge, MA, USA), SRP9 and SRP14 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), SRP54 and SRP68 (Proteintech, Chicago, IL, USA), SRP72 (Novus, Littleton, CO, USA), c-tubulin and b-actin (Boster, Wuhan, China).

Quantitative Real-time RT-PCR
Total RNA was prepared using TRIzolH reagent (Invitrogen) and incubated with RNase-free DNase I (Fermentas, Glen Burnie, MD, USA) for 30 min. The DNA-free RNA was reverse transcribed using a M-MLV reverse transcription kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Samples prepared in the absence of reverse transcriptase served as negative controls. SYBR green real-time PCR was performed using the ABI PRISM 7300 Sequence Detection system (Applied Biosystems). The level of GAPDH mRNA served as an internal control. Primer sequences used to detect Dicer and GAPDH mRNAs were as described [17]. Primer sequences (forward and reverse) for SRP proteins were as follows:

Fractionation of Postnuclear Extracts to Analyze SRP Assembly
Fractionation of postnuclear extracts was carried out as described with modifications [21]. The HepG2.2.15 cells were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 25 mM KCl, 5 mM MgCl 2 , 0.01% octaethylene glycol dodecyl ether (Nikko, Tokyo, Japan), 1 mM dithiothreitol, 30 U/ ml RNase inhibitors (Promega), 0.25% Triton X-100, and a cocktail of protease inhibitors). The homogenized cells were incubated at 0uC for 10 min, potassium acetate was added to 500 mM, and nuclei were isolated by centrifuging the homogenate at 8006g for 10 min. The postnuclear fraction (1 mg) was incubated with 30 pmol 7SL RNA fragments or control LacZ RNA at 0uC for 10 min, 37uC for 60 min, layered onto a 10-40% glycerol gradient, and centrifuged at 5uC for 8 h at 40,000 rpm in a Beckman SW41Ti rotor. Ten equal fractions were collected from the top to the bottom of the gradient. One-third of each fraction was used for RNA extraction using TRIzol LS (Invitrogen), and the level of 7SL RNA was measured by real-time RT-PCR using the following primers, 59-GGAGTTCTGGGCTG-TAGTGC-39 and 59-ATCAGCACGGGAGTTTTGAC-39 to specifically detect the full-length 7SL RNA. The remainder of each fraction was treated with trichloroacetic acid to precipitate proteins for western blot analysis.

Detection of Cell Surface Glycoproteins
Cells were washed three-times with phosphate-buffered saline (PBS) and stained with Alexa FluorH 488 conjugate-lectin GS-II (100 mg/ml, Invitrogen) at room temperature for 30 min. The cells were then washed three-times and resuspended in PBS. Fluorescence was detected using FACSAria (BD Biosciences) and analyzed using WinMDI software (http://facs.scripps.edu/ software.html).

ECFP-ER Expression
HEK293T cells were transfected with the plasmid pECFP-ER (Invitrogen). This plasmid encodes enhanced cyan fluorescent protein (ECFP) fused at its 59 and 39 ends to the endoplasmic reticulum (ER) targeting sequence of calreticulin and the ER retrieval sequence, KDEL, respectively. A cell line stably expressing pECFP-ER was transfected with specific RNAs as indicated, and images of fluorescent cells were obtained using a Leica DMI3000 B microscope. When compared with the corresponding control, the images were taken with the same light intensity. However, to present the results more clearly, different light intensities were applied in different experiments.

Assays for Secreted Alkaline Phosphatase (SEAP)
Cells were co-transfected with a plasmid encoding SEAP (pSEAP2-control, BD Biosciences, San Jose, CA, USA) and different RNAs or shRNAs as indicated. The growth medium was changed every 24 h and collected at indicated time points. SEAP activity was determined using the Great EscAPe SEAP kit (BD Biosciences) following the manufacturer's instructions.
Cloning of the Dicer-processed 7SL RNA Fragments

Statistical Analysis
Each experiment was independently performed at least three times. All data are shown as mean 6 SD except as specifically indicated. Statistical significance was determined by Student's 2tailed t-test, and results were considered significant at p,0.05 when compared with the control.

Results
The 7SL RNA-Derived sRNA5cd does not Modulate SRP-Mediated Protein Targeting 7SL sRNA5cd accounts for more than 95% of the ,20 nt 7SL RNA-derived small RNAs [16]. To address whether Dicerprocessed 7SL RNA fragments modulate the efficiency of SRPmediated protein targeting, we first investigated the effect of 7SL sRNA5cd on the secretion of SEAP [22]. Co-transfection of the pSEAP2-control plasmid and different concentrations of 7SL sRNA5cd indicated that 7SL sRNA5cd did not significantly affect the secretion of SEAP (Fig. S1A). We then monitored the expression of a specific reporter protein ECFP-ER, which is an enhanced cyan fluorescent protein fused to the ER-targeting sequence of calreticulin. The fluorescence intensities in 7SL sRNA5cd-transfected cells were comparable to those in control small RNA-transfected cells (Fig. S1B). Finally, we measured the expression of cell surface glycoproteins in cells transfected with 7SL sRNA5cd or control small RNA. Our results indicated that transfection of 7SL sRNA5cd did not alter the expression of total cell surface glycoproteins (Fig. S1C). Taken together, these results indicate that 7SL sRNA5cd did not modulate the efficiency of SRP-mediated protein targeting. 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) We then tested whether these 7SL RNA fragments interfere with the formation of the SRP complex. Postnuclear extracts incubated with the control RNA or 7SL RNA fragments were fractionated on glycerol gradients. As shown in Fig. 2, after incubation with the control RNA, most SRP54 and SRP68 proteins migrated in fractions 5 to 7 together with 7SL RNA. These SRP subunits and the full-length 7SL RNA shifted to the upper fractions after treatment with the synthetic 7SL RNA fragments, suggesting that part of the SRP complex may dissociate and hence migrate at lower sedimentation rates.

Dicer Knockdown Enhances SRP-Mediated Protein Targeting
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.
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 Dicerknockdown 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).
Dicer is essential for the biogenesis of microRNAs that may target SRP subunits, thus decreased Dicer expression may result in elevated levels of SRP proteins. To exclude the possibility that the increased efficiency of ER-mediated protein targeting in Dicer knockdown cells was mediated by increased levels of SRP proteins, the expression of SRP subunits was monitored using real-time RT-PCR and western blotting analyses. We found that knockdown of Dicer did not alter the expression of SRP proteins (Fig. S8).

Discussion
We previously demonstrated that Dicer processes 7SL RNA into fragments of different lengths [16]. In the present study, we demonstrate that several 7SL RNA fragments interfered with the formation of the SRP complex (Fig. 2), and thus inhibited the expression of ECFP-ER and cell surface glycoproteins as well as the secretion of SEAP (Fig. 1). Furthermore, knockdown of Dicer resulted in increased expression of ECFP-ER and cell surface glycoproteins and enhanced secretion of SEAP (Fig. 3), which were partially restored by transfection with the 7SL RNA fragment mixture (Fig. 4). Taken together, our data suggest that Dicer regulates SRP function via the following mechanism: Dicer processes a proportion of 7SL RNA into fragments of different lengths. Some of the 7SL RNA fragments function as dominantnegative regulators of the full-length 7SL RNA, interfering with the formation of the SRP complex and inhibiting its function.
Decreased Dicer expression reduces the levels of the 7SL RNA fragments, thereby enhancing SRP-mediated protein targeting, which eventually increases the expression of cell surface glycoproteins and SEAP secretion.
Of note, 7SL RNA fragments longer than 200 nt or lacking the 7SL sRNA5cd sequence cannot be detected by our cloning method. Another limitation of this method is that any RNA shorter than 200 nt that contains a sequence similar to that of 7SL sRNA5cd can be cloned. Therefore, further effort is necessary to develop a more specific and efficient method to clone all of the 7SL RNA fragments.

Conclusions
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 that interfere with the function of the full-length 7SL RNA.