To assess whether the different 5′-exons have different expression patterns, PCRs were carried out on mouse brain cDNA at different stages from E11 to E18 and at postnatal stages from P0 to adult. We used forward primers in the alternative first exons and reverse primers in exons 2 and 5 (Fig.3 A
). As shown in Fig.3 B
, exons 1A and 1D were expressed in all stages tested. Amplification of exon 1C was weak, indicating low level expression in the tissues examined (data not shown). The complex UTR 1B was barely detectable at E11 and E12, whereas two main bands were amplified in RNA isolated from brain at E15 and later, including adult (Fig.3 B
). We tested the expression of the alternative first exons in P19 cells, which differentiate into neurons in the presence of RA (Fig. 3 C
). Exons 1A and 1D were detected in undifferentiated and differentiated P19 cells. In contrast, the expression of UTR 1B was complex. Multiple bands were amplified in undifferentiated cells and up to 4 days after RA induction. When neural induction was complete, only two bands were visible. This developmental regulation was confirmedin vivo
. As shown in Fig. 3
), a pattern of multiple bands amplified from E11 mouse RNA becomes restricted to two main amplicons at P0 and adult. Sequencing of the two main amplicons showed that they are formed of fragments 1B1, 1B2, and 1B4, with alternative inclusion of fragment 1B8. Interestingly, the sequences of fragments 1B1, 1B2, and 1B4 are conserved in mouse, man, and macaque. Sequencing of the other amplicons confirmed that they correspond to different combinations of exons 1B1–1B10, as shown in Fig. 3 E
. These fragments contain variable numbers of ATG codons, suggesting that some uORFs are excluded from segment 1B in parallel to neuronal differentiation (Fig. 3 E
and Table II
). The ability of uORFs to down-regulate translation is documented in mammals, as mentioned above (
- van der Velden A.W.
- Thomas A.A.
). Although the brain is the major site of Dab1
expression, low mRNA levels are also detected in the kidney, liver, and uterus (
- Howell B.W.
- Gertler F.B.
- Cooper J.A.
). By RT-PCR, exons 1A and 1D were amplified from the brain, testis, kidney, and liver, but not from the spleen, heart, or thymus. By contrast, the mature forms of exon 1B were solely amplified from brain cDNA, confirming its neuron-specific expression in the adult.
Northern blot analysis was performed using probes corresponding to the different alternative first exons and a probe covering the PTB domain-encoding region as a control. With the PTB domain probe, three bands of ∼5.5, 4, and 1.3 kb were revealed in poly(A) RNAs from E17 and P0 brain and correspond to the pattern described previously (
- Howell B.W.
- Gertler F.B.
- Cooper J.A.
- Ware M.L.
- Fox J.W.
- Gonzalez J.L.
- Davis N.M.
- Lambert de Rouvroit C.
- Russo C.J.
- Chua Jr., S.C.
- Goffinet A.M.
- Walsh C.A.
). Satisfactory results were achieved with the exon 1A probe, which revealed three bands with similar sizes and an additional band of ∼1.8 kb (Fig. 4
). No signal could be detected with a probe for exon 1D. A probe for UTR 1B revealed a major 5.5-kb band and several smaller and fainter bands, all of which are absent in RNA extracted from scrambler
mouse brain (data not shown). No clear correspondence between the bands revealed on Northern blots and the alternative forms of the Dab1
RNA could be established.
We analyzed the expression of the different 5′-first exons by in situ
hybridization with 33
P-labeled riboprobes. The short sequence of the exons and their high GC content (∼75% for exons 1A and 1D) made it difficult to obtain a signal, and satisfactory results could be obtained only with the complex UTR 1B. As shown in Fig. 5
), the UTR 1B expression pattern at E14 and in the newborn (P0) brain was very similar to that observed with the PTB probe (A–C
), with the exception that the ventricular zone appeared to be very weakly labeled at both stages. Both signals were also similar in other parts of the brain. This suggests that 5′-UTR 1B and the form containing exon 1A are the two major Dab1
forms in the brain. This was confirmed using multiplex RT-PCR on adult brain RNA reactions, which yielded approximate proportions of messages containing exons 1A, 1B, and 1D in the brain of 47, 33, and 20%, respectively.