ABSTRACT
Normal septation of the cardiac outflow tract requires migration of neural crest cells from the posterior rhombencephalon to the branchial arches and developing conotruncal endocardial cushions. Proper migration of these cells is mediated by a variety of molecular cues. Adhesion molecules, such as integrins, are involved in the interaction of neural crest cells with the extracellular matrix, while cadherins allow neural crest cells to interact with each other during their migration. Pax3 appears to be important for proliferation of neural crest precursors, and connexin-43-mediated gap junction communication influences the rate of migration. Endothelin and its receptors are required for normal postmigratory differentiation. Platelet-derived growth factor and retinoic acid have roles in neural crest migration and differentiation as well. Finally, the similarity between the cardiovascular malformations seen in the DiGeorge and 22q11 deletion syndromes and animal models of neural crest deficiency has led to the examination of the role of genes located near or within the DiGeorge critical region in neural crest migration.
Subject(s)
Cell Movement/physiology , Neural Crest/cytology , Animals , Aorta/abnormalities , Aorta/embryology , Cell Adhesion Molecules/physiology , Cell Movement/genetics , Chick Embryo , Chromosomes, Human, Pair 22/genetics , Chromosomes, Human, Pair 22/ultrastructure , Connexin 43/physiology , DNA-Binding Proteins/physiology , DiGeorge Syndrome/embryology , DiGeorge Syndrome/genetics , Endothelin-1/deficiency , Endothelin-1/genetics , Endothelin-1/physiology , Extracellular Matrix Proteins/physiology , Gap Junctions/physiology , Gap Junctions/ultrastructure , Growth Substances/physiology , Heart/embryology , Humans , Mice , Mice, Knockout , Mice, Mutant Strains , Neural Tube Defects/genetics , Neural Tube Defects/metabolism , Neurotrophin 3/physiology , PAX3 Transcription Factor , Paired Box Transcription Factors , Receptors, Growth Factor/physiology , Receptors, Retinoic Acid/physiology , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.
Subject(s)
RNA Splicing , RNA, Messenger/metabolism , Adenoviruses, Human/genetics , Base Sequence , In Vitro Techniques , Molecular Sequence Data , Nucleic Acid Precursors/metabolism , Oligodeoxyribonucleotides/chemistry , RNA, Messenger/ultrastructure , RNA, Viral/metabolism , Stereoisomerism , Sulfur/chemistryABSTRACT
Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.
