Abstract
BackgroundThe genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.Methodology/Principal FindingsWe have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.SignificanceOur observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.
Highlights
Hox genes play a crucial role in specifying regional identity along the body axes and in regulating morphogenesis during animal development
Molecular characterization of the Hoxa5 alternate transcripts Previous northern analysis of polyA+ RNA from mouse embryo using a DNA probe corresponding to the 39-untranslated region of the second exon of the Hoxa5 gene has shown that polyadenylated transcripts of approximately 1.8, 5.0, 9.5 and 11.0 kb in length contain sequences from the Hoxa5 locus [23]
We applied a series of molecular approaches, and by merging all the data obtained from northern, 39- and 59-Rapid Amplification of cDNA ends (RACE), RT-PCR and cDNA analyses, we established a schematic representation of the major Hoxa5 transcripts produced in the e12.5 mouse embryo (Fig. 1)
Summary
Hox genes play a crucial role in specifying regional identity along the body axes and in regulating morphogenesis during animal development. The clustered organization appears fundamental for the precise spatio-temporal regulation and the function of each Hox gene and for the correct patterning of the embryo. Multiple overlapping transcriptional units exist at the Hoxa locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation
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