Figure 3.

Function of SLM proteins in Nrxn1, Nrxn2, and Nrxn3 alternative splicing regulation in vitro. (A) Splice reporters for Nrxn1, Nrxn2, and Nrxn3 AS4. Nrxn splice reporter constructs contain AS4 with constitutive exons (dark gray), alternative exon 20 (light gray), and introns shown as lines. Intron sizes are indicated (drawings not to scale). (B) Splice reporter assays with various RNA-binding proteins. Reporter expression vectors were cotransfected into HEK293T cells with epitope-tagged expression constructs for GFP-SAM68, GFP-SLM1, GFP-SLM2, YFP-hnRNPA1, or YFP-hnRNPH1. Alternative splice isoform choice was measured by semi-quantitative RT-PCR with primers flanking the alternatively spliced segment. Fragment sizes for AS4(+) and AS4(−) isoforms are 318 bp and 228 bp (Nrxn1), 270 bp and 180 bp (Nrxn2), and 354 bp and 264 bp (Nrxn3). Expression of transfected RNA-binding proteins was confirmed by Western blotting with anti-GFP antibodies. (C) Dose-dependent activity of STAR family proteins toward Nrxn, Nrxn2, and Nrxn3 AS4. Splice reporter processing was assessed in experiments with increasing amounts of DNA encoding RNA-binding proteins transfected (DNA amounts used indicated in micrograms). (D) Alternative splicing was assayed by semi-quantitative RT-PCR. Expression levels of GFP-tagged proteins were detected by immunoblot. (E) Schematic drawing of domain organization of SAM68 and SLM1 and chimeric mutants proteins. An HA-tag was attached to the C-terminal end of each open reading frame. (F) Splice reporter assays with chimeric proteins using Nrxn1 and Nrxn2 AS4 reporters. Alternative splicing was assayed by semi-quantitative RT-PCR. Expression of the HA-tagged proteins was confirmed by immunoblotting.