FIG. 4.

Inclusion of exon 4 in HeLa cells is repressed by truncated TIAR lacking RRM domains. (A) Diagrams of wild-type TIAR (TIAR.FL) and a truncated form of TIAR (TIAR.Q) that contains the carboxy-terminal Q-rich domain but lacks the RRM domains. (B) HeLa cells that preferentially include exon 4 were cotransfected with a calcitonin/CGRP reporter and increasing amounts of either the TIAR.FL or the TIAR.Q plasmid. The left panel shows a Western blot produced by using tag-specific antibodies (anti-HA antibody) to document production of the desired TIAR forms following transfection (lanes 1 to 4). The middle panel shows Western blot analysis with anti-TIAR antibody to indicate the level of the overexpressed TIAR protein relative to the level of endogenous TIAR. The two bands represent the alternatively spliced isoforms of TIAR (3). The right panel shows results of an RT-PCR assay of total RNA from transfections of HeLa cells with the wild-type calcitonin/CGRP reporter gene and no TIAR (lane 1) or increasing amounts of full-length TIAR (FL; lanes 2 and 3) or truncated TIAR (Q; lanes 4 and 5). Products and percentages of inclusion are indicated as in Fig. 1B and D. (C) Results of RT-PCR assay of total RNA from HeLa cells transfected with the calcitonin/CGRP reporter carrying mutations at the pseudo 5′ splice site or with wild-type U1 snRNA (wt; lane 1) or suppressor U1 snRNA containing compensatory mutations (mt; lanes 2 to 4) and either no TIAR (lanes 1 and 2) or TIAR.Q (lane 3) or TIAR.FL (lane 4) plasmid.