Figure 3

TIA proteins bind to the human β-actin mRNA 3′-UTR. (A) TIA and HuR proteins interact with β-actin mRNA. HeLa extracts were incubated with anti-TIA1 (in the absence or presence of RNase A), anti-TIAR, anti-HuR and anti-U2AF65 antibodies. Immunoprecipitated RNAs were isolated and quantified the β-actin mRNA levels by semiquantitative RT-PCR. The resulting PCR products were analyzed by 2% agarose-electrophoresis. In lane 1 (indicated as c above legend), 1/10 of the HeLa extract used in immunoprecipitation assays was amplified by RT-PCR. As a negative control, the beads were used (lane 7). (B) RNA map of TIA proteins on human β-actin (ACTB) gene by iCLIP analysis. Crosslinking sites of TIA1 and TIAR on the human β-actin gene in HeLa cells. The bar graph shows the number of cDNAs that identifies each crosslinking site [9]. Note that the highest density of crosslinking sites of the TIA1 and TIAR proteins is located at the 3′-UTR of the β-actin mRNA (for more details see Additional file 2: Figure S2). (C) Nucleotide sequence of the 3′-UTR of the human β-actin mRNA (NM_001101.3). The full-length sequence of the β-actin 3′-UTR was divided to generate four probes. In the probes 1 and 3, the major sites bind TIA proteinswere underlined. (D) RNA band shift assays of TIA proteins to β-actin 3′-UTR. Electrophoretic mobility shift assays were performed incubating GST (500 ng), GST-TIA1, GST-TIAR and MBP-HuR (0, 10, 100, 500 and 1000 ng) proteins with ³²P-labeled β-actin 3′-UTR RNA. (E and F) Ultraviolet crosslinking assays illustrating the direct interaction between TIA1, TIAR or HuR proteins and β-actin 3′-UTR RNA probes. The F and P1-P4 legends correspond to full-length β-actin 3′-UTR RNA and the four β-actin RNA probes indicated in C, respectively. Molecular weight markers and the identities of protein-RNA complexes are shown.