eIF4AII is dispensable for miRNA-mediated gene silencing

(A) Generation of NIH/3T3 eIF4AII^em1JP cells. Schematic diagram illustrating the exon/intron structure of the murine eIF4AII gene and denoting exon 5 (orange shading; downward arrow) targeted by Cas9 for deletion mutagenesis. The positions of the primers used to characterize the eIF4AII locus following CRISPR/Cas9-mediated gene editing are shown. The red sequence denotes the region to which the Cas9 sgRNA was designed and the underlined 5′-GGG-3′ triplet indicates the position of the PAM motif. The orange highlighted sequence denotes a duplicated region that likely led to microhomology-mediated NHEJ to generate Δ1 (see text for details). The TPGR motif 1b is shown in green. (B) PCR analysis of eIF4AII^em1JP. DNA was isolated from parental NIH/3T3 cells (lane 1), NIH/3T3 cells expressing Cas9 and an sgRNA targeting the neutral Rosa26 locus (Malina et al. 2013) (lane 2), or eIF4AII^em1JPcells (lane 3) and used in PCRs with the indicated primer pairs (Table 1). PCR products were analyzed on 1% agarose gels. Products were excised from the gel and analyzed by direct sequencing. (C) Schematic diagram representing configuration of the Δ1 eIF4AII allele in eIF4AII^em1JP. (D) Schematic diagram representing configuration of the Δ2 eIF4AII allele in eIF4AII^em1JP. (E) RT-PCR analysis of mRNA isolated from the indicated cell lines. Complementary DNA was generated as indicated in Materials and Methods using an eIF4AII-specific primer. PCRs were performed using the indicated primer pairs and products analyzed on a 1.2% agarose gel. Products were excised from the gel and analyzed by direct sequencing. (F) RT-qPCR analysis of eIF4AII mRNA in NIH/3T3 and eIF4AII^em1JP cells. Expression of eIF4AII was calculated relative to GAPDH levels and is based on ΔΔCT values. (G) Western blot probing of extracts prepared from NIH/3T3 (lane 1) or eIF4AII^em1JP (lane 2) using antibodies directed to the proteins shown to the right. (H) Western blot probing of extracts prepared from NIH/3T3 (lane 1) or eIF4AII^em1JP (lane 2) using an N-terminal directed antibody (ab31218, Abcam). Arrow indicates position of migration of eIF4AII.