Research Associate RI-MUHC MONTREAL, Quebec, Canada
Abstract Body : Mutations in a common core spliceosomal factor called SNRPB causes cerebrocostomandibular syndrome (CCMS). Most CCMS patients have point mutations that increase levels of transcripts containing a pre-termination codon (PTC) containing alternative exon 2 (AE2). Herein, we generated a mouse line with a 61-base pair intronic deletion upstream of AE2 (D61) and show that 10% of heterozygous and homozygous embryos (Snrpb D61/+;Snrpb D61/D61) had abnormalities similar to those found in CCMS patients. D61 mutants had microcephaly, defects in the bones of the craniofacial region and ribs and 18/47 die from 4 weeks of age onwards. These embryos also had a significant increase in expression of the AE2 and a reduction in Snrpb levels. In parallel, we generated a conditional mutant mouse carrying loxp sequences flanking exons 2–3 of Snrpb. We used mesoderm-specific Mesp1-Cre to delete Snrpb, and showed that a fraction (5/40) of heterozygous Snrpbloxp/+; Mesp1-Cre+/- embryos are abnormal at E9.5. They have a narrow frontonasal prominence, a smaller 2nd pharyngeal arch, an enlarged heart, and begin to die at E12.5 where 50% are found alive. In these mutants, Snrpb expression was not changed whereas expression of the AE2 was half of controls, suggesting a potential compensatory increase of the Snrpb wild-type allele. To test if the D61 mutation fails to complement Mesp1-Cre mediated deletion of Snrpb, we generated Snrpbloxp/D61; Mesp1-Cre+/- mutant embryos. At E9.5, all of the recovered double heterozygous embryos showed an unlooped heart, misshapen somites and failed to turn. These embryos had a significant reduction in Snrpb levels without a change in AE2 expression. Our findings suggest that the 61-bp intronic region regulates AE2 inclusion and plays an important role in Snrpb regulation. Thus, these sequences should be investigated in CCMS patients that do not carry mutations in SNRPB coding exons.
