GH1 Splicing Is Regulated by Multiple Enhancers Whose Mutation Produces a Dominant-Negative GH Isoform That Can Be Degraded by Allele-Specific Small Interfering RNA (siRNA)

Abstract

The majority of mutations that cause isolated GH deficiency type II affect splicing of GH1 transcripts, leading to the production of a dominant-negative GH isoform. Because numerous mutations and polymorphisms throughout the GH1 gene have not yet been tested for aberrant splicing, we used a deletion mutagenesis screen across intron 2-exon 3-intron 3 to identify splicing regulatory sequences. These analyses identified a new enhancer element, ESE2, upstream of the cryptic splice site in exon 3 and further defined a previously described enhancer (ESE1) to include the first seven nucleotides of exon 3. Besides enhancers, the overall size of intron 3 is also crucial for exon inclusion. Given the deleterious effects of the dominant-negative 17.5-kDa isoform, these and previous studies underscore the extent to which splicing regulatory elements serve to prevent exon skipping. Importantly, we show here that small interfering RNAs can be used to specifically degrade exon 3-skipped transcripts, potentially a new avenue of therapeutic intervention in isolated GH deficiency II and other dominant disorders.

Association of single nucleotide polymorphisms in interferon signaling pathway genes and interferon-stimulated genes with the response to interferon therapy for chronic hepatitis C

“…When the sequence of rs3213545 changed to allele T, as ctctTgt, this specific region was no longer recognized as SF2/AFF consensus ESE site. Mutation within an ESEs has been reported to result in reduction of the level of full-length transcripts and the corresponding proteins [27]. Thus, it is likely that T allele carriers of this SNP may have lower expression of OASL as compared with C allele carriers.…”

siRNA therapeutics: big potential from small RNAs

“…This targeting approach was used to degrade specifically transcripts encoding the 17.5 kDa isoform with no apparent effect on either the 22kDa isoform or another, smaller 20 kDa isoform. 24 Thus, properly designed siRNAs can be used to specifically degrade aberrant or alternatively spliced mRNAs (Figure 2c and d).…”

A novel splicing mutation in exon 4 (456G>A) of the GH1 gene in a patient with congenital isolated growth hormone deficiency

“…16,[27][28][29] The overall size of intron 3 of the GH1 gene has been shown to be crucial for exon 3 inclusion. 30 Two dominant negative splicing mutations have been described in intron 2 of the GH1 gene, causing complete skipping of exon 3 from the GH1 mRNA transcript. IVS2 -2A>T mutation, first revealed in a IGHD II family from the region of central Russia, affects the highly conserved tagGAA sequence of the invariant agent of the 3'-acceptor splice site in intron 2.…”

“…Exon splice enhancer element (ESE1) includes the first seven nucleotides of exon 3. 30 A heterozygous G-to-T transversion at the first nucleotide of the exon 3 (E3 +1G>T) deleted exon 3 in mature mRNA and resulted in IGHD II in a Japanese family. 34 E3 +5A>G mutation has also been described.…”