Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line

Abstract

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Fig. 1. Representation of SPC2 mRNA and δRz

A, the organization of SPC2 includes the signal peptide (S), the prodomain (Pro), the catalytic domain, and the P-domain. B, secondary structure and nucleotide sequence of the δRz hybridized to the SPC2 mRNA. The pseudoknot P1.1 is illustrated by the dotted lines. The homopurine bp at the top of the P4 stem is represented by two large dots (G●●G) whereas the wobble bp is represented by a single large dot (G●U). Only nucleotides essential for cleavage are shown: H indicates A, C, or U, and N indicates A, C, G, or U. The arrow indicates the cleavage site.

Fig. 2. Autoradiograms of 5% PAGE gels of both the RNase H and δRz cleavage assays

A and B, typical results of RNase H and δRz assays, respectively. The 1st lanes are the negative controls without either ODN or δRz. The other 8 lanes are the assays performed with either the various ODNs or δRz. The number after the ODN and δRz indicates the cleavage site position on SPC2 mRNA. The asterisks indicate the lower intensity cleavage bands. The molecular sizes (nt) are indicated on the right.

Fig. 3. Expression of tRNA^Val-δRz chimeric constructions

A, vector used for cultured cells studies. We attenuated the CMV promoter of pcDNA3.1/hygro by the cleavage of MfeI before the promoter sequence. MfeI and EcoRI are two compatible sites that have been used for the cloning. The δRz has been first cloned in the pPUR-KE vector. Then the tRNA^Val-δRz cassette has been subcloned from pPUR-KE to pcDNA3.1/hygro without CMV promoter to obtain ptRNA^Val-δRz-SPC2-X (where X indicates the position of the cleavage site). The hygromycin gene resistance allowed selection of positive clones upon transfection. B, drawing of the chimeric tRNA^Val-δRz RNA produced from the ptRNA^Val-δRz-SPC2 hybridized with the SPC2 mRNA substrate. The tRNA^Val portion drives the δRz to the cytoplasm, and the poly(U) ensures the transcription termination. C, analysis of the expression levels of the chimeric tRNA^Val-δRz and U6 RNA. Primer extension of the chimeric constructions tRNA^Val-δRz (δRz line) on total RNA of four cell lines established with either ptRNA^Val-δRz-SPC2–154 (154-1 and 154-2) or -394 (394-1 and 394-9). Untransfected βTC-3 and cell line established with the vector ptRNA^Val-KE (βTC-3 and KE, respectively) served as controls. Primer extension of the endogenous U6 RNA (U6 line) served as internal control.

Fig. 4. Autoradiograms of Northern blot hybridizations

A, analysis of SPC2 mRNA is presented in the upper panel. The levels of SPC2 mRNA were analyzed in the five established cell lines. The lower panel illustrates the relative expression of SPC2 mRNA by densitometric analysis (using the KE cell line as a reference; n = 3). Each band was normalized with the corresponding 18 S ribosomal RNA band. B–E, analysis of SPC1, SPC3, SPC7, and CPE mRNAs levels, respectively. The size markers are shown on the right.

Fig. 5. Electron microscopy of βTC-3 and 154 cell lines (A and B, respectively)

Secretory granules are indicated by arrows (×25,000).

Fig. 6. Western blot hybridizations

Upper panel, proteins corresponding to the pro- and mature SPC2 enzymes (75 and 68 kDa, respectively) are shown. Lower panel, major bands (50 –56 kDa) corresponding to CPE are represented. Lanes are as follows: βTC3 cells, untransfected control cell line; KE, transfected with vector without the ribozyme minigene; 154, transfected with SPC2-ribozyme; 154C47A, transfected with SPC2-ribozyme with a point mutation; 394, transfected with SPC2-ribozyme (inaccessible cleavage site); 394C47A, transfected with same ribozyme as 394 with a point mutation.

Fig. 7. RP-HPLC analysis of cellular extracts for their Dyn A-(1–8) IR content

A, schematic representation of pro-Dyn processing by SPC2, SPC3, and CPE producing either α-neo-endorphin, Dyn A-(1–17), Dyn B-(1–13), C-peptide, Dyn A-(1–9), or Dyn A-(1–8). In the inset, basic amino acids found at the cleavage sites of Dyn A-(1–17) as shown. Total protein extracts from untransfected βTC-3, 394, and 154 cell lines were submitted to RP-HPLC (B–D, respectively). 50 μg of total proteins was injected for βTC-3 and 394-1 and 0.5 mg for cell line 154. The values are expressed on the ordinate as femtomoles per RIA tube. The gradient used for the RP-HPLC analysis is indicated (dashed lines) as percentage of ACN (ordinate axis on the right). The arrows indicate the elution position of a standard peptide (14 min, 22% ACN).

Fig. 8. Typical MALDI-time of flight mass spectra

These spectra were recorded in the positive linear delayed extraction mode of HPLC collected fractions eluted at 35 and 31% (A and B, respectively) of control cell line. Peaks absent for the 154 cell line fraction analysis are illustrated in red.