Alternative pre-mRNA processing of the calcitonin/CGRP gene. (A) Schematic diagram of the calcitonin/CGRP gene and its alternative RNA processing in thyroid and neuronal cells. (B) Diagram showing the calcitonin/CGRP reporter gene and its cell-specific RNA processing patterns. (C) Left, RT-PCR assay of total RNA from HeLa or CA77 (rat medullary thyroid carcinoma) cells transfected with the diagramed calcitonin/CGRP reporter gene. Amplification bands resulting from exon 4 inclusion or exclusion are indicated. Right, a picture of CA77 cells.

Hu proteins interact with the U-tract in the intronic element. (A) RNAs used in the UV cross-linking/IP and RNA gel mobility shift assay. Wild-type and mutated U-tract sequences are indicated. (B) UV cross-linking/IP assay. The ³²P-labeled in vitro transcribed RNAs were UV cross-linked in CA77 cell nuclear extract and immunoprecipitated with antibodies specific to Hu proteins. (C) RNA gel mobility shift assay. The ³²P-labeled in vitro transcribed RNAs were incubated with 0, 1, 2, 10, and 50 ng of GST-HuB.

Targeted binding of TIA-1 to the U-tract region increased exon 4 inclusion. (A) Diagram showing the location of the RRE stem-loop sequence. (B) RT-PCR analysis using RNAs isolated from transfected CA77 cells that preferentially exclude exon 4. CA77 cells were transfected with a RRE-containing reporter (lanes 1–5) in the absence of any protein plasmids (lanes 1) or the presence of 1 and 2 μg of Rev (lanes 2 and 3) or TIA-1-Rev (lanes 4 and 5). (C) RT-PCR analysis using RNAs isolated from CA77 cells transfected with the RRE-containing reporter in the absence of any proteins (lane 1) or the presence of 0.4 μg of Rev (lane 2) or TIA-1-Rev (lanes 3–9) together with 0.2–0.4 μg of HuC (lanes 4 and 5), Rev (lanes 6 and 7) or HuC-Rev (lanes 8 and 9). The bottom panel in B and C shows a bar graph indicating the level of the calcitonin exon inclusion upon overexpression of the transfected proteins.

Association of Hu proteins and the calcitonin/CGRP pre-mRNA in CA77 cells. The nuclear extract prepared from CA77 cells was immunoprecipitated with anti-Hu sera. RNA was isolated from either total nuclear extract or immunoprecipitated pellet and analyzed by RT-PCR. Minus RT lanes are included as controls. The oligonucleotides used to amplify the calcitonin/CGRP pre-mRNA anneal to sequences in intron 4, and the resulting PCR product is 225 nnucleotides, whereas the oligonucleotides used to amplify the GAPDH pre-mRNA anneal to sequences in intron 3 and exon 5, and the resulting PCR product is 585 nucleotides.

Expression of Hu proteins correlates with the CGRP processing pathway. (A) Hu proteins are highly expressed in cells that undergo CGRP-specific RNA processing. Western blot analysis was carried out with human anti-Hu serum or anti-TIA-1/R antibody. Splicing pathways were determined by RT-PCR of total RNA isolated from these cells with (for cells that do not express the gene endogenously) or without transfection of the calcitonin/CGRP reporter gene. HeLa and CHO cells have been used as cell models for the calcitonin pathway, whereas F9 (mouse teratocarcinoma) and PC12 (rat pheochromocytoma) cells have been used as cell models for the CGRP pathway. CA77 (rat) and TT (human) cells are derived from medullary thyroid carcinomas, and SK-N-SH is a human neuroblastoma cell line. One-sixth of total proteins was loaded in the lanes F9 and mouse ES in the top panel. RT-PCR analysis of RNA isolated from cells that express the calcitonin/CGRP gene was shown. The calcitonin product is 308 nucleotides for rat and mouse and 276 nucleotides for human. The CGRP product is 284 nucleotides for rat and mouse and 237 nucleotides for human. (B) Correlation between expression of neuron-specific Hu proteins (HuB, HuC, and HuD) and the neuronal RNA processing phenotype. A calcitonin/CGRP reporter gene was transfected with untreated RA-induced or DMSO-induced mouse P19 cells. Total RNA was isolated from the transfected cells, and semiquantitative RT-PCR was carried out using primers specific for mouse HuA, HuB, HuC, HuD, GAPDH, or the calcitonin/CGRP reporter gene. Amplification bands resulting from exon 4 inclusion (nonneuronal) or exclusion (neuronal) are indicated. The percentage of inclusion of exon 4 is indicated below each lane.

Hu proteins compete with TIAR for binding at the U-tract sequence. (A) Gel mobility shift assay. The ³²P-labeled in vitro transcribed RNAs indicated in Figure 3A were incubated with GST-TIAR and His-HuD. Lane 1, no protein added; lanes 2–5, 0.1 μg of His-HuD and 0, 0.02, 0.2, and 2 μg of GST-TIAR were added; and lanes 6–9, 0.1 μg of GST-TIAR and 0, 0.02, 0.2, and 2 μg of His-HuD were added. (B) UV cross-linking/IP assay. Top, ³²P-labeled in vitro-transcribed RNAs was UV cross-linked in CA77 cell nuclear extract in the absence (lane 1) or presence of 0.2, 1, and 5 μg of GST-TIAR (lanes 2–4) and immunoprecipitated with antibodies specific to Hu proteins (anti-Hu sera) and TIA-1/TIAR (3E6). Bottom, ³²P-labeled in vitro-transcribed RNAs were UV cross-linked in HeLa cell nuclear extract in the absence (lane 1) or presence of 0.1, 0.5, and 2.5 μg of GST-mHuC (lanes 2–3) and immunoprecipitated with antibodies specific to Hu proteins (anti-Hu sera) and TIA-1/TIAR (3E6). (C) Reduced binding of TIA-1/TIAR to the U-tract sequence in CA77 cells. Left, Western blot analysis using same amounts of nuclear proteins from HeLa and CA77 cells and anti-TIA-1/TIAR antibody. Right, UV cross-linking/IP assay using ³²P-labeled RNA substrate indicated in Figure 3A, same nuclear protein amounts of HeLa and CA77 cell nuclear extracts, and anti-TIA-1/TIAR antibody.

Neuronal exclusion of exon 4 in CA77 cells is compromised by a truncated mHuC protein containing RRM3 domain with the hinge domain. (A) Diagram of the two truncated forms of mHuC is shown in the top panel. (B) CA77 cells were cotransfected with a calcitonin/CGRP reporter and increasing amounts (2 and 4 μg) of either mHuC RRM12 or mHuC RRM3 plasmid. The top panel shows an RT-PCR assay of total RNA from transfections of CA77 cells with the wild-type calcitonin/CGRP reporter gene and no mHuC (lane 1), increasing amount of mHuC RRM12 (lanes 2 and 3), or mHuC RRM3 (lanes 4 and 5). The middle panel shows Western blot analysis by using anti-Xpress antibody to indicate the level of the overexpressed mHuC proteins in HeLa cells. The bottom panel shows a bar graph indicating the level of the calcitonin exon inclusion upon overexpression of the transfected proteins.