FIGURE 3. The hnRNP L high affinity CA repeat element in CaMKIV- and hnRNP L-regulated splicing and its location dependence.

A, the CA repeat element (or CG mutant as shown in sequence) is transferred to different positions of the middle exon or flanking introns in splicing reporters L2-L5 to replace the corresponding sequences of the vector DUP175 (except in reporter L2, the element was inserted upstream the branch point as indicated). Positions of the first nucleotide of the elements are indicated above the exon or introns, relative to the last nucleotide of upstream intron, first nucleotide of exon, or downstream intron, respectively. For comparison, the relative positions of L1 and L1m elements in DUP175ST are also indicated. B, an agarose gel of the RT-PCR products of the splicing reporters with co-expressed CaMKIVm, CaMKIV, or protein kinase A (PKA) in HEK293T cells, with average percentages (±S.D.) of exon inclusion under each lane and exon-included or -excluded products indicated to the right. NC, PCR negative control. *, same as in Fig. 2. C, effect of hnRNP L overexpression on splicing reporters L1-L5. Western blots (upper panel) of overexpressed hnRNP L-FLAG (anti-FLAG tag) and His-YB-1 (anti-His tag) from HEK293T cells cotransfected with reporters L1-L5 or mutants and His-YB-1 (Y) or hnRNP L-FLAG (L) plasmids. *, a nonspecific band recognized by the anti-His tag antibody. An agarose gel (lower panel) of RT-PCR products of the splicing reporters with the overexpressed hnRNP L or YB-1, similarly labeled as in B.