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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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In vitro transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases (A: lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled 32P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold (B).
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Figure 2: In vitro transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases (A: lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled 32P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold (B).

Mentions: Preliminary in silico analyses obtained using the mFold program (41,42) suggested that the ATM Δ pseudoexon sequence could fold upon itself to form a very compact, double-stranded structure (Figure 2B). Its existence was thus tested experimentally by partial RNA digestion using single and double-strand-specific RNAses such as V1 (which cleaves double-stranded RNA and stacked RNA regions), T1 (which cleaves single-stranded guanosines) and S1 nuclease (which cleaves single-stranded RNA without sequence specificity) (Figure 2A). The overall position of the different cleavages shows that the ATM Δ pseudoexon sequence does indeed display a structure that is consistent with the model proposed in Figure 2B. In addition, it is interesting to note that two particularly strong RT stops (indicated by arrows in Figure 2A) seem to enclose the pseudoexon sequence, possibly reflecting the difficulty of the RT to travel across a compact structure.Figure 2.


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)

In vitro transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases (A: lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled 32P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: In vitro transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases (A: lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled 32P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold (B).
Mentions: Preliminary in silico analyses obtained using the mFold program (41,42) suggested that the ATM Δ pseudoexon sequence could fold upon itself to form a very compact, double-stranded structure (Figure 2B). Its existence was thus tested experimentally by partial RNA digestion using single and double-strand-specific RNAses such as V1 (which cleaves double-stranded RNA and stacked RNA regions), T1 (which cleaves single-stranded guanosines) and S1 nuclease (which cleaves single-stranded RNA without sequence specificity) (Figure 2A). The overall position of the different cleavages shows that the ATM Δ pseudoexon sequence does indeed display a structure that is consistent with the model proposed in Figure 2B. In addition, it is interesting to note that two particularly strong RT stops (indicated by arrows in Figure 2A) seem to enclose the pseudoexon sequence, possibly reflecting the difficulty of the RT to travel across a compact structure.Figure 2.

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