Novel stage-specific alternative splicing switches in erythroid genes. (A) General scheme for detection of alternative splicing of a pre-mRNA (left) into the mRNA isoform including the alternative exon (top right) or the mRNA skipping this exon (bottom right). Diagnostic isoform-specific microarray probes are indicated above the spliced mRNAs, whereas PCR primers used for validation are shown below the mRNAs (arrows). In addition, there are exon probes for the first and third exons that hybridize equally to both isoforms and are useful for determining overall transcript levels. (B) Shown are gels of PCR products validating alternative splicing switches in late erythropoiesis (left) and diagrams of the relevant pre-mRNA regions (right). Gels demonstrate substantial increases in exon inclusion products (top bands, indicated by arrows), relative to exon-skipping products (bottom bands), at day 14. The deduced splicing patterns are indicated at the right, with black lines indicating major splice patterns at day 7 and red lines indicating predominant splice pattern at day 14. Asterisks indicate positions of stop codons (not shown for ARFIP1 and PLD1 because they are located farther downstream). Common names of the alternatively spliced transcripts are as follows: HNRPLL indicates heterogeneous nuclear ribonucleoprotein L-like (an hnRNP protein); SNRP70, U1 small nuclear ribonucleoprotein 70K (a component of the U1 snRNP); MBNL2, muscleblind 2 (RNA binding proteins with known splicing regulatory activity); ATP11C, ATPase class VI type 11C; ARFIP1, ADP-ribosylation factor interacting protein 1; PLD1, phospholipase D1.