Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

Abstract

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level.

Microduplications at the pseudoautosomalSHOXlocus in autism spectrum disorders and related neurodevelopmental conditions

“…SHOXa is expressed in numerous tissues, including skeletal muscle, placenta, heart and bone marrow fibroblasts, among others; SHOXb expression is more restricted and is predominantly found in fetal kidney, skeletal muscle and bone marrow fibroblasts 28. Durand et al 29 also reported SHOXa and SHOXb expression in different fetal and adult human brain regions, including fetal hindbrain, thalamus and basal ganglia, and adult thalamus, cerebellum and frontal cortex, pointing to an additional function of SHOX during fetal brain development and maintenance of brain function. The same study also identified novel SHOX exons (2a, 7–1, 7–2 and 7–3), which create new coding and untranslated regions, resulting in at least four novel SHOX splice variants.…”

“…Furthermore, the genomic region downstream of SHOX2 harbours a highly conserved non-coding sequence homologous to the SHOX enhancer CNE5, which has been found to function primarily as a neural enhancer 36. Intriguingly, expression analysis in chicken embryos has revealed that Shox and Shox2 expression patterns overlap only partially in the developing chicken limb, whereas Shox expression is completely covered by Shox2 expression in the developing chicken brain 29. Consistent with these findings, it has been speculated that SHOX2 may partly take over the functions of SHOX in the neural tissues of cases with SHOX haploinsufficiency, mitigating the effects on neurodevelopment 29…”

“…Pseudoautosomal region 1 (PAR1) coordinates are according to chromosome X, GRCh37/hg19 assembly. Information about genes and transcript isoforms in the region (in blue) was obtained from the RefSeq database and supplemented with the novel SHOX transcripts identified by Durand et al (in purple) 29. Upstream and downstream evolutionarily conserved non-coding elements (CNEs) acting as SHOX enhancers are depicted in red 30–35.…”

Copy number variants in patients with short stature

“…43 Case II.5, with a terminal de novo 15q deletion located 1.5 Mb downstream of IGF1R and 244 kb downstream of the ADAMTS17 locus on the reverse strand, had a normal birth size, but showed proportionate progressive growth failure (SH/H ratio þ 1.58 SDS) with a normal head circumference. Clinical characteristics included slight frontal bossing of the skull, a high pitched voice and slight abdominal adiposity and delayed bone age.…”

In-Silico Algorithms for the Screening of Possible microRNA Binding Sites and Their Interactions

“…Three recently published articles on miRNA regulations describe the utilization of miRWalk and TargetScan predictions in copy number variants [56], different splice variants of a gene [57] and single nucleotide polymorphisms (SNP) [53]. …”