Ectopically expressed shRNAs and synthetic siRNAs, but not ectopically expressed microRNAs are able to interfere with each other and with the endogenous microRNA pathway through their ability to be incorporated into RISC. However, the relative strength of competition can be equalized for all the tested siRNA sequences by reducing the cellular concentration of the HIV-1 TAR RNA binding protein (TRBP). Thus, our results suggest that TRBP is a sensor in the loading of the RNA guide sequence into RISC. MATERIALS AND METHODS ShRNAs and MicroRNAs constructions U6shRNA-expressing constructs were synthesized via a PCR-based reaction that includes primers encoding the shRNA with a 30 region complementary to the U6 promoter and a primer complementary to the 50 end of the U6 promoter as described previously (25). All PCR reactions were carried out as follows: 1 min at 948C, 1 min at 558C and 1 min at 728C for 30 cycles. The resulting PCR cassettes were cloned directly into the pCR2.1 plasmid (TA cloning vector, Invitrogen). The shRNA constructs used for the competition experiments are targeted against sequences present in the HIV-rev (shSII and shSI) HIV tat (shSI, shTAT), HIV vif (shVif) and the EnvPb1 retrovirus envelope (shL) genes. The shRNA Luc (Luc) is targeted to a sequence present in the coding region of the ﬁreﬂy luciferase gene. All shRNAs are expressed by transcription from the U6 promoter. The synthetic siRNAs (siH3, siH6 and siH1) are targeted against sequences present in the coding region of the hnRNPH gene. Refer to the Supplementary Information for speciﬁc sequences for the shRNAs and siRNAs. All other oligonucleotide sequences are available upon request. The microRNA constructs were similarly constructed using overlapping primers containing the wild type or a variant mir30 sequence. The mir30 guide sequence was replaced with 21 nt complementary to a site present in the HIV-rev transcript (SII) by including the corresponding nucleotide mutations in the PCR primers. The resulting PCR products were cloned into pcDNA3.1 under the control of the CMV promoter. A cassette containing the U1 promoter, the ﬁreﬂy luciferase coding sequence, and the SV40 polyadenylation signal was digested with EcoRI and DraII and cloned into the Bluescript vector (Stratagene) to create the ﬁreﬂy luciferase target (FLT). The Renilla luciferase target (RLT) was generated by cloning a 21-nt HIV-rev sequence (SII) into the XhoI site located in the 30-untranslated region (30-UTR) of the humanized Renilla luciferase gene of plasmid psiCHECK-2TM (Promega). Cell lines and transfection conditions Cells were grown in DMEM (Irvine Scientiﬁc, Santa Ana, CA, USA) supplemented with 10% fetal calf serum (Irvine Scientiﬁc), 1 mM L-glutamine. To generate the HCT116-GFPmiR21 cell line, HCT116 cells were trans- fected (using Lipofectamine 2000, Invitrogen) with SapI Nucleic Acids Research, 2007, Vol. 35, No. 15 5155 linearized pcDNA4/GFPmir21 plasmid DNA (4) (gen- erous gift from Thomas Tuschl) and cells with stable plasmid integrations were selected by applying 200 mg Zeocin per ml media for 10 days. Single colonies were picked and screened for expression of EGFP upon transfection with a 20O-methyl RNA complementary to miR21 relative to a control 20O-methyl RNA complemen- tary to miR33. For the competition experiments, 293 cells were transfected with the shRNA expression constructs and the corresponding targets in 24-well plates using Lipofectamine PlusTM reagent (Life Technologies, GibcoBRL) as described by the manufacturer. One hundred nanogram of the FLT plasmid was co-transfected with 12.5, 25, 50 or 100 ng of the shRNA-expressing constructs and 0.2 ng of the Renilla luciferase plasmid which was included to normalize for transfection eﬃciency for each reaction. The synthetic siRNAs were transfected to achieve 10 nanomolar concentrations in all co- transfection experiments. However, 100 nanomolar con- centrations of each siRNA were used for transfection in the HCT116-GFPmiR21 cell line. One hundred nanomo- lar concentrations of the 20O-Me oligonucleotides were also used for transfection in this cell line. One hundred nanogram of the microRNA constructs were co-transfected with 20, 50 or 100 ng of RTL and yielded the same relative results in each case. Here, 0.5 ng of ﬁreﬂy luciferase were used to normalize for transfection eﬃciency in each sample. However, 800 ng of both the shRNAs and the microRNAs were used for the transfec- tions in the HCT116-GFPmiR21 cell line. For the overexpression of Exportin-5, 100 ng of an Exportin-5 expression plasmid (kindly provided by Thomas Tuschl) was co-transfected with 100 ng of the FLT plasmid and 50 ng of each shRNA construct. Ten (or) twenty nanomolar of each synthetic siRNA were also used for this experiments and yielded analogous results. The TRBP down-regulation was achieved by transfect- ing 40 nM of anti-TRBP siRNAs (26) (siTRBP-A siTRBP-B and siTRBP1; sequences are available upon request) in a 100 mm plate of 293 cells. Cells were transfected in suspension at 50% conﬂuency using the siQuest transfection reagent (Mirus, Madison, WI, USA) as suggested by the manufacturer. The following day cells reached 70% conﬂuency and were transfected a second time using the siQuest transfection reagent. Twenty-four hours after the second transfection, cells were lifted, seeded in a 24-well plate, and transfected with the various constructs as described in the Results section. Cells were lysed 24 h after this ﬁnal transfection and processed for luciferase assays. Each experimental sample was normalized against its own target control, which included the corresponding target co-transfected with one or more irrelevant controls. For example, down-regulation of the FLT by the U6 anti-luciferase shRNA (Luc) was calculated by comparing the luciferase down-regulation obtained with the Luc- speciﬁc shRNA to the luciferase units obtained when the target was co-transfected with an irrelevant shRNA. When speciﬁc shRNAs were co-transfected with