Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. We created site-directed mutants at either adjacent splice sites that were analyzed alone or in the presence of complementary U1 snRNAs. The cryptic ATM exon is shown as a grey box with dotted outline. In the affected cells, amplification with the ex19F and in20R primers revealed a pre-mRNA form that corresponds to exon 19, exon 20, the cryptic exon and retention of the downstream portion of intron 20 Figure 5A , lane 1 , whereas analysis with in20F and ex22R did not show any intermediate Figure 5A , lane 2. In this construct, the splicing intermediate lacking the preceding intron is embedded in the natural context with the flanking ATM exon 20, downstream intron sequences, and ATM exon The position where the ATM intron 20 sequences were cloned is indicated.

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To further evaluate the role of deep intronic elements in splicing regulation, we compared human and mouse intron 20 sequences in the region of the ISPE. However, not all intronic mutations create or strengthen splice sites, suggesting the existence of yet unknown splicing regulatory elements 89.

Intronic sequences distant from the canonical splice sites are mostly considered functionally neutral regarding pre-mRNA splicing and are thus rarely considered when mapping splicing regulatory elements and are not routinely sequenced in human genetics screening.

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All amplified bands were verified by sequencing. In this paper, we have carefully characterized the composition of cis -acting sequences around the ISPE signal and the role of non-canonical U1 snRNP binding.

To analyze the interference with the cryptic splice sites more in detail, we identified the pre-mRNA splicing precursors derived from lymphoblast cells and from hybrid minigene experiments. In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge.


Altogether, the data show that while in the wild-type situation no intron precursors can be detected, in the ISPE deletion, accumulation of precursors retaining the downstream intron can be seen.

X-linked hypophosphatemia attributable to pseudoexons of the PHEX gene. Mouse ATM minigenes were transfected in Hep3B cells and the pattern of splicing analyzed with specific primers.

True genes for human U1 small nuclear RNA. RNA was extracted 48 h later and cDNA was prepared using random primers and reverse transcriptase as previously described Thus, it is possible that the non-canonical interaction of U1 snRNP at the ISPE might affect intron splicing efficiency by regulating the polymerase II processivity or elongation rate due to the qtm association between transcription and splicing 35 In order to induce aberrant splicing, deletion of the mouse-conserved ISPE requires at least the presence of cryptic splice sites that define the boundaries of the exon Figure 3 suggesting that additional cis -acting elements adjacent sasy the ISPE are involved.

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A novel mutation in the cystic qtm gene in patients with pulmonary disease but normal sweat chloride concentrations. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: Above each exon are depicted the modified U1 snRNAs binding sites.

The same PCR conditions were applied for the other minigene transfections using the following specific primers: This position-dependent effect indicates that, as previously suggested for normal exons 22binding of U1 snRNP inside the cryptic exon might produce steric interference between complexes involved in the recognition of splicing signals, and in this case, the acceptor splice site. In fact, exonic splicing enhancers have been identified not only in alternatively spliced exons, but also in constitutive globin exons Lanes 1 and 3 correspond to amplification with ex19F and in20R, lanes 2 and 4 to amplification with in20F and ex22R.

Ataxia-telangiectasia is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition 10 — Open in a separate window. Even if this evidence seems to exclude that the U1 snRNP complex formed on deep ISPE participates in resplicing, the precursors originating from the usage of the resplicing signal might be rapidly processed to the mature mRNA and are not detected in the amplification assay.


Hereditary Neuropathies and Spinocerebellar Atrophie. This reduction in exon definition may not be simply due to weak splice sites, but result from more complex exon-specific interactions that involve both exonic and intronic regulatory elements whose function strongly depend on the context of individual exons. It is possible that the weaker exon definition present in the alternative spliced EDA and CF cryptic mutation, as compared with the constitutive globin exon, mediates the splicing inhibitory effect of the U1 snRNAs.

Promoter architecture modulates CFTR exon 9 skipping. Transfection of these mutants induced aberrant splicing with inclusion of the cryptic exon resembling the splicing pattern observed with the original GTAA ISPE deletion found in the affected patient Figure 2B.

Functional studies on the ATM intronic splicing processing element

Total RNA was amplified using primers specific for each construct. The lower and higher MW bands correspond to normal intron processing and inclusion of the 65 bp cryptic exon, respectively. Control experiments, in which the same U1 snRNAs were transfected along with the WT minigene, did not show changes in the splicing pattern data not shown. CblE type of homocystinuria due to methionine synthase reductase deficiency: Genomic variants in exons and introns: A point mutation in the human estrogen receptor gene is associated with the expression of an abnormal estrogen receptor mRNA containing a 69 novel nucleotide insertion.

Abstract In disease-associated genes, eassy understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge.