Novel methodologies to generate small RNA expression and small RNA sponges lentiviral libraries Miguel A. Moreno-Mateos, Cristina Rodriguez-Mateo, Jose A. Pintor-Toro Avda. Americo Vespucio s/n. Edif. CABIMER Parque Cientifico y Tecnolgico Cartuja 93 CP Sevilla (SPAIN) Telephone: Conclusion The work presented here provides a detailed description regarding the generation of libraries of small RNAs and anti-small RNAs or small RNA sponges. One the one hand, our data highlight the power of the system to generate novel small RNA expression libraries that can be used to perform large-scale screens for gain of function phenotypes in any given biological phenomenon. As a proof of principle, and as an example of the identification of novel functions for known miRNAs, we isolated two miRNAs that confer cellular resistance to anoikis. Furthermore, the unbiased nature of our technology provides the possibility to discover previously undescribed miRNAs and even other types of small RNAs with regulatory function, which constitutes an important breakthrough when compared to current approaches. On the other hand, the small RNA sponge library is based on novel and original technologies that will enable us to block any small RNA present into the cell. This new technology has notable advantages; among them, it is accessible to any laboratory with some training in molecular biology, it is relatively cheap and its execution is non-dependent of any sophisticated infrastructure. So far, there are no anti-small RNA libraries to carry out functional genetic screenings, and, therefore, they show an unexplored potential for high-throughput screenings related to many biological processes and pathologies. We believe that this methodology will help to the better understanding of the complex mechanisms underlying the control of gene expression by small non-coding RNAs. Figure 1. Mature miRNAs expressed under the control of dual convergent promoters are functionally similar to full-length expressed miRNAs. (A) Schematic drawing showing the plasmid used to express mature miRNAs and others small RNAs. (B) RNAse protection assay of miR-21, miR-19a transiently overexpressed from the pLENT-DUAL plasmid (upper and lower panel). miR-21 full-length (miR-21 FL) was expressed from a pCMV-miR-21 plasmid. Control (Ctr) cells expressed the empty vectors pLENT-DUAL or pCMV. Y corresponds to yeast RNA. (C) Transient transfections were carried out in HEK-293T cells, using pLENT-DUAL-21 (expressing the mature form of miR-21) or pCMV-miR-21 full-length, pLuc-BS carrying a binding site for miR-21, pCMV-Renilla (normalization control) at 100:10:1 proportions, respectively. Firefly and Renilla activities were measured 40 h after transfection (D) Similar luciferase assay as described in C using a plasmid harbouring a 652 nt wild-type sequence corresponding to the 3′-UTR of Pdcd4 at the 3′-position of the luciferase reporter gene. (E) Similar luciferase assay as described in C using HCT116 Dicer -/- cells infected with a lentivirus expressing a small hairpin RNA against Dgcr8 were used. All transfections were performed in triplicate, and results are shown as the averages ± standard errors of the means. The data were subjected to two-tailed student’s t test (* p< 0.05; ** p< 0.01; *** p< 0.001) 3’utr pdcd4 ** A) B) C) D) E) Novel small RNA expression libraries uncover hsa-miR-30b/c as key factors in anoikis resistance Figure 2. A functional screening using a novel small RNAs expression library reveals hsa-miR-30b and hsa-miR-30c as repressors of anoikis. (A) Scheme illustrating the small RNA cloning strategy to generate expression libraries based on the pLENT-DUAL lentiviral vector. (B) Pie chart representing the percentage of the different class of small RNAs in the library. On the right, a list of 15 high frequency miRNAs in the library. Below, a correlation between miRNAs expressed in MDA-MB-231 cells and miRNAs cloned in the library (logarithmic scale). (C) Immortalized RPE1 cells were infected with a lentiviral expression library containing a small RNAs collection from the MDA-MB-231 cell line. RPE1 infected cells were cultured for 19 days in agar dishes at low confluence avoiding any contact and surviving clones transferred to adhesive culture dishes to allow the growth of anoikis resistant cells. DNA from these clones was isolated and sequenced. (D) Table showing the small RNA sequences overexpressed in anoikis resistant clones. (E) Percentage of apoptotic RPE1 cells (SubG1) infected with lentivirus containing candidate mature miRNAs or empty vector (control). Three days after infection, cells were deprived of serum for 24 hours and then cultured in polyhema (PH) plates plus methyl cellulose (2%) in serum free medium for another 24 hours. (F) Percentage of apoptosis in RPE1 cells infected with lentivirus carrying full-length miR-30b, miR-30c-1, miR-30b/c mature miRNAs (Lent-30b/c) and the corresponding empty vectors as controls in a similar assay as described in E. The experiments were performed in triplicate and results are shown as the averages ± standard errors of the means. The data were subjected to two-tailed student’s t test (* p< 0.05; ** p< 0.01) B) Full-lengthpLENT-DUAL * * ** C) D)E) * ** Ctr 30b 30c Active CASP3 ACTB Pro-CASP3 p53 ACTB Ctr 30b 30c ACTB BIM Ctr 30b 30c *** ** A)B)C) Figure 3. Hsa-miR-30b and hsa-miR-30c downregulate CASP3 expression through its 3’ UTR for anoikis resistance. (A) Western blot analysis of p53, CASP3 and BCL2L11 (BIM), three putative target proteins of miR-30b/c related to anoikis. RPE1 cells were infected with lentivirus expressing full-length miR-30b, miR-30c-1 (miR-30c) or empty vector as control. Active CASP3 was analysed in cells cultured under anoikis conditions as described in Figure 2E.  -Actin (ACTB) was used as the loading control. (B) Schematic representation of the caspase-3 3’UTR containing two putative binding sites for miR-30b/c. The 3’UTR of caspase-3 mRNA was cloned downstream of the open reading frame of luciferase (pLuc-BS). Both broadly ( ) and poorly ( ) conserved putative miRNA regulatory elements of the caspase-3 3’UTR were mutated (Mut1 and Mut2, respectively) and cloned along with the wild type 3’UTR (WT). HEK-293T cells were transiently contransfected with plasmids overexpressing full-length miRNAs (miR-30b, miR-30c-1 or empty vector as control), pLuc-3’UTR CASP3 (WT, Mut1 or Mut2) and pCMV-Renilla at 100:10:1 proportions, respectively. Firefly and Renilla activities were measured 40 h after transfection. The luciferase activity normalized to Renilla is shown (lower panel). Transfections were performed in triplicate, and results are shown as the average ± standard errors of the means The data were subjected to two-tailed student’s t test (** p< 0.01; *** p< 0.001) (C) RPE1 cells were co-infected with both lentiviruses expressing full-length miR-30b/c and the ORF of caspase-3. Western blot analysis of pro-CASP3, active CASP3 and ACTB is shown for RPE1 cells growing either in complete-medium under adherent conditions (-PH) or in anoikis-inducing conditions (+PH). Flow citometry analysis of the levels of apoptosis in CASP3 infected cells growing under anoikis-inducing conditions (lower panel). Figure 4. Hsa-miR-30b and hsa-miR-30c reduce cell death induced by TRAIL and delay acini clearance in MCF10A cells. (A) Cells were infected with lentivirus containing full-length miR-30b, miR-30c and empty vector as control. Four days after infection, cells were cultured in the presence of Trail (500 ng/ml) for seven hours and the percentage of apoptotic cells was determined (SubG1). (B) Effects of overexpression of miR-30b/c on MCF10 acini morphogenesis assays. Cells were infected as in A and assayed for acinar clearance. For 24 days, acini were fixed and analyzed by confocal microscopy every 6 days. The experiment was performed in triplicate, and 100 acini were analyzed in each condition. Percentage of acini containing four or less intact nuclei in lumen was measured at the indicated time points during morphogenesis. Results are shown as the averages ± standard errors of the means. The data were subjected to two-tailed student’s t test (*p< 0.05; ** p< 0.01) A)B) Design and generation of small RNA sponges libraries A)B) Figure 5. Small RNA sponges library. (A) Schematic drawing of the strategy to generate a miRNA sponge library. S, sense small RNA; AS, antisense small RNA (B) Map of the pSIN-DUAL vector. Luciferase gene has been inserted and two restriction sites, NotI and XbaI, have been generated into the 3’ UTR for subcloning small RNAs sponges. Abstract MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNAs libraries carrying out genetic screens by gain-of-function and lack-of-function approaches have been used to uncover their different roles. However, these strategies have shown limitations due to their high cost and/or technical difficulties. In this work, we have developed two novel methodologies to express or deplete mature miRNAs and other small RNAs. With the first methodology we have generated lentiviral-based libraries expressing small RNAs, and with the second methodology we have generated lentiviral-based libraries of anti-small (sponge) RNAs. Both libraries contain the whole small RNA population from any given cell type or tissue including known and unknown small RNAs. This opens the possibility of identifying novel small RNA-coding genes with a physiological role. Altogether, these novel methodologies provide powerful and effective ways to reveal novel small RNAs involved in a particular biological process. U6H1 uu 5’ pLENT-DUAL uu 5’ A) Small RNA isolation nt MDA-MB-231 (4175) 5’P5’P OH 3’ 5’P5’P ddC 3’ 5’OH ddC 3’ T4 RNA ligase ddC 3’ RT-PCR 5’OH OH 3’ BbvII pLENT-DUAL U6 H1 5’OHOH 3’ dephosphorylation phosphorylation 5’P5’P ddC 3’ T4 RNA ligase Cloning Independent clones selection Culture in attached conditions CCTACAGGTA Anoikis screening (19 days in agar) Sequencing surviving clones sRNA candidates F) Patent pending: P