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RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events.

Buratti E, Dhir A, Lewandowska MA, Baralle FE - Nucleic Acids Res. (2007)

Bottom Line: However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations.Our results indicate that RNA structure is a major splicing regulatory factor in both cases.Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

ABSTRACT
Genomic variations deep in the intronic regions of pre-mRNA molecules are increasingly reported to affect splicing events. However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations. In this work we have examined the structural architecture of pseudoexons previously described in ATM and CFTR patients. The ATM case derives from the deletion of a repressor element and is characterized by an aberrant 5'ss selection despite the presence of better alternatives. The CFTR pseudoexon instead derives from the creation of a new 5'ss that is used while a nearby pre-existing donor-like sequence is never selected. Our results indicate that RNA structure is a major splicing regulatory factor in both cases. Furthermore, manipulation of the original RNA structures can lead to pseudoexon inclusion following the exposure of unused 5'ss already present in their wild-type intronic sequences and prevented to be recognized because of their location in RNA stem structures. Our data show that intrinsic structural features of introns must be taken into account to understand the mechanism of pseudoexon activation in genetic diseases. Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

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Related in: MedlinePlus

(A) A schematic diagram of the two deletion mutants (CFTR Del1 and CFTR Del2) inserted in the PY7 system starting from the CFTR MUT plasmid. The splicing pattern displayed by these mutants either in vitro (left panel) or when transfected in Hep3B cells (right panel) is shown in (B). (C) The schematic diagram of an additional set of CFTR mutants (CFTR Dis and CFTR Rep) that were subjected to in vitro splicing (D: left panel) and transfected in Hep3B cells (D, right panel).
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Figure 7: (A) A schematic diagram of the two deletion mutants (CFTR Del1 and CFTR Del2) inserted in the PY7 system starting from the CFTR MUT plasmid. The splicing pattern displayed by these mutants either in vitro (left panel) or when transfected in Hep3B cells (right panel) is shown in (B). (C) The schematic diagram of an additional set of CFTR mutants (CFTR Dis and CFTR Rep) that were subjected to in vitro splicing (D: left panel) and transfected in Hep3B cells (D, right panel).

Mentions: Indeed, disruption of the major structural features of this RNA region should affect the efficiency of CFTR pseudoexon inclusion. Thus, we began testing this by selectively deleting the supporting lower stem in either of the two strands (mutants CFTR Del1 and Del2, see Figure 7A for a schematic diagram). In parallel, in order to test the importance of sequence conservation within the pseudoexon the structure was either ‘disrupted’ and then ‘repaired’ by engineering mutants CFTR Dis and CFTR Rep (see Figure 7C for a schematic diagram). These mutants were then tested either in vitro or following transfection in Hep3B cells for ability to recognize the pseudoexon (Figure 7B and D, respectively).


RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events.

Buratti E, Dhir A, Lewandowska MA, Baralle FE - Nucleic Acids Res. (2007)

(A) A schematic diagram of the two deletion mutants (CFTR Del1 and CFTR Del2) inserted in the PY7 system starting from the CFTR MUT plasmid. The splicing pattern displayed by these mutants either in vitro (left panel) or when transfected in Hep3B cells (right panel) is shown in (B). (C) The schematic diagram of an additional set of CFTR mutants (CFTR Dis and CFTR Rep) that were subjected to in vitro splicing (D: left panel) and transfected in Hep3B cells (D, right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935003&req=5

Figure 7: (A) A schematic diagram of the two deletion mutants (CFTR Del1 and CFTR Del2) inserted in the PY7 system starting from the CFTR MUT plasmid. The splicing pattern displayed by these mutants either in vitro (left panel) or when transfected in Hep3B cells (right panel) is shown in (B). (C) The schematic diagram of an additional set of CFTR mutants (CFTR Dis and CFTR Rep) that were subjected to in vitro splicing (D: left panel) and transfected in Hep3B cells (D, right panel).
Mentions: Indeed, disruption of the major structural features of this RNA region should affect the efficiency of CFTR pseudoexon inclusion. Thus, we began testing this by selectively deleting the supporting lower stem in either of the two strands (mutants CFTR Del1 and Del2, see Figure 7A for a schematic diagram). In parallel, in order to test the importance of sequence conservation within the pseudoexon the structure was either ‘disrupted’ and then ‘repaired’ by engineering mutants CFTR Dis and CFTR Rep (see Figure 7C for a schematic diagram). These mutants were then tested either in vitro or following transfection in Hep3B cells for ability to recognize the pseudoexon (Figure 7B and D, respectively).

Bottom Line: However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations.Our results indicate that RNA structure is a major splicing regulatory factor in both cases.Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

ABSTRACT
Genomic variations deep in the intronic regions of pre-mRNA molecules are increasingly reported to affect splicing events. However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations. In this work we have examined the structural architecture of pseudoexons previously described in ATM and CFTR patients. The ATM case derives from the deletion of a repressor element and is characterized by an aberrant 5'ss selection despite the presence of better alternatives. The CFTR pseudoexon instead derives from the creation of a new 5'ss that is used while a nearby pre-existing donor-like sequence is never selected. Our results indicate that RNA structure is a major splicing regulatory factor in both cases. Furthermore, manipulation of the original RNA structures can lead to pseudoexon inclusion following the exposure of unused 5'ss already present in their wild-type intronic sequences and prevented to be recognized because of their location in RNA stem structures. Our data show that intrinsic structural features of introns must be taken into account to understand the mechanism of pseudoexon activation in genetic diseases. Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

Show MeSH
Related in: MedlinePlus