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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 ATM WT construct. White boxes represent tropomyosin exons 2 and 3 whilst the single line represents the intron. The arrows indicate the primers used for the RT-PCR analyses of the processed RNA templates. The ATM intronic region is in small letters whilst pseudoexon sequences are in bold capital letters. The ISPE sequence is underlined and the GTAA nucleotides deleted in the ATM Δ construct are in italic. Within the ATM sequence all potential donor and acceptor splice-site sequences together with their scores calculated according to the NNSplice program are underlined (ND indicates that the splice site is not detected as a viable donor site by the program). It should be noted that although the ISPE sequence scores very well as a potential donor site it is never used by the splicing machinery. (B) In vitro splicing analysis of the ATM WT and ATM Δ RNAs both using radioactive templates (upper panel) and by RT-PCR (lower panel). In order to investigate 5′int/5′pe donor-site usage a number of mutants were engineered in the ATM Δ construct (C shows a schematic diagram). In mutant ATM 46-48T Δ the 46A and 48G nucleotides were replaced by a T in the ATM Δ context. This improved 5′int donor site was then inactivated by introducing an A for a G substitution in position 41 (mutant ATM 46-48T 41A Δ). Cryptic 5′int donor-site improvement was also obtained by deleting the G in position 41 (mutant ATM Δ G41). (D) In vitro splicing analysis of these mutants.
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Figure 1: (A) A schematic diagram of the ATM WT construct. White boxes represent tropomyosin exons 2 and 3 whilst the single line represents the intron. The arrows indicate the primers used for the RT-PCR analyses of the processed RNA templates. The ATM intronic region is in small letters whilst pseudoexon sequences are in bold capital letters. The ISPE sequence is underlined and the GTAA nucleotides deleted in the ATM Δ construct are in italic. Within the ATM sequence all potential donor and acceptor splice-site sequences together with their scores calculated according to the NNSplice program are underlined (ND indicates that the splice site is not detected as a viable donor site by the program). It should be noted that although the ISPE sequence scores very well as a potential donor site it is never used by the splicing machinery. (B) In vitro splicing analysis of the ATM WT and ATM Δ RNAs both using radioactive templates (upper panel) and by RT-PCR (lower panel). In order to investigate 5′int/5′pe donor-site usage a number of mutants were engineered in the ATM Δ construct (C shows a schematic diagram). In mutant ATM 46-48T Δ the 46A and 48G nucleotides were replaced by a T in the ATM Δ context. This improved 5′int donor site was then inactivated by introducing an A for a G substitution in position 41 (mutant ATM 46-48T 41A Δ). Cryptic 5′int donor-site improvement was also obtained by deleting the G in position 41 (mutant ATM Δ G41). (D) In vitro splicing analysis of these mutants.

Mentions: The PY7 plasmid has been described in detail elsewhere (38). For our purposes, we have inserted two SmaI and NdeI unique cloning sites at positions 44 and 50, respectively, in its 111-nt long intron. Both the wild-type (wt) and gtaa-deleted (Δ) ATM sequences were amplified from the pATM plasmids used in the original report (32) and inserted in the SmaI site of PY7 (Figure 1A). The oligos used for this task were the following: 5′-ttgctcaagctcttaactgcaacagtggt-3′ (s) and 5′-gtcaaacagaaaattcaaatcccag-3′ (as). In order to obtain the ATM 46-48T Δ mutant a two-step PCR method was used with the following primers 5′-tgagggtacgtatgccctagatg-3′ (s) and 5′-catctagggcatacgtaccctca-3′ (as). From this mutant we obtained ATM 46-48T 41A Δ using primers 5′-cactctactgatgaggatacg-3′ (s) and 5′-cgtatcctcatcagtagagtg-3′ (as). Finally, the primers used to insert the 21–23 substitution in both the ATM Δ and ATM 46-48T Δ mutants were the following: 5′-gtgatataccctcactctac-3′ (s) and 5′-gtagagtgagggtatatcac-3′ (as). On the other hand, to obtain the ATM 5′-new Δ mutant we created a unique StuI site by joining together nucleotides 38–40 (agg) with nucleotides 51–53 (cct). The ATM 5′-new Δ mutation was then introduced by ligating in this site the 5′-gtaggtaagtacgaaggc-3′ (s) and 5′-gccttcgtacttacctac-3′ (as) oligos. Donor splice-site scores were calculated according to the NNSplice 0.9 program available at http://www.fruitfly.org/seq_tools/splice.html (39).Figure 1.


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 ATM WT construct. White boxes represent tropomyosin exons 2 and 3 whilst the single line represents the intron. The arrows indicate the primers used for the RT-PCR analyses of the processed RNA templates. The ATM intronic region is in small letters whilst pseudoexon sequences are in bold capital letters. The ISPE sequence is underlined and the GTAA nucleotides deleted in the ATM Δ construct are in italic. Within the ATM sequence all potential donor and acceptor splice-site sequences together with their scores calculated according to the NNSplice program are underlined (ND indicates that the splice site is not detected as a viable donor site by the program). It should be noted that although the ISPE sequence scores very well as a potential donor site it is never used by the splicing machinery. (B) In vitro splicing analysis of the ATM WT and ATM Δ RNAs both using radioactive templates (upper panel) and by RT-PCR (lower panel). In order to investigate 5′int/5′pe donor-site usage a number of mutants were engineered in the ATM Δ construct (C shows a schematic diagram). In mutant ATM 46-48T Δ the 46A and 48G nucleotides were replaced by a T in the ATM Δ context. This improved 5′int donor site was then inactivated by introducing an A for a G substitution in position 41 (mutant ATM 46-48T 41A Δ). Cryptic 5′int donor-site improvement was also obtained by deleting the G in position 41 (mutant ATM Δ G41). (D) In vitro splicing analysis of these mutants.
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Figure 1: (A) A schematic diagram of the ATM WT construct. White boxes represent tropomyosin exons 2 and 3 whilst the single line represents the intron. The arrows indicate the primers used for the RT-PCR analyses of the processed RNA templates. The ATM intronic region is in small letters whilst pseudoexon sequences are in bold capital letters. The ISPE sequence is underlined and the GTAA nucleotides deleted in the ATM Δ construct are in italic. Within the ATM sequence all potential donor and acceptor splice-site sequences together with their scores calculated according to the NNSplice program are underlined (ND indicates that the splice site is not detected as a viable donor site by the program). It should be noted that although the ISPE sequence scores very well as a potential donor site it is never used by the splicing machinery. (B) In vitro splicing analysis of the ATM WT and ATM Δ RNAs both using radioactive templates (upper panel) and by RT-PCR (lower panel). In order to investigate 5′int/5′pe donor-site usage a number of mutants were engineered in the ATM Δ construct (C shows a schematic diagram). In mutant ATM 46-48T Δ the 46A and 48G nucleotides were replaced by a T in the ATM Δ context. This improved 5′int donor site was then inactivated by introducing an A for a G substitution in position 41 (mutant ATM 46-48T 41A Δ). Cryptic 5′int donor-site improvement was also obtained by deleting the G in position 41 (mutant ATM Δ G41). (D) In vitro splicing analysis of these mutants.
Mentions: The PY7 plasmid has been described in detail elsewhere (38). For our purposes, we have inserted two SmaI and NdeI unique cloning sites at positions 44 and 50, respectively, in its 111-nt long intron. Both the wild-type (wt) and gtaa-deleted (Δ) ATM sequences were amplified from the pATM plasmids used in the original report (32) and inserted in the SmaI site of PY7 (Figure 1A). The oligos used for this task were the following: 5′-ttgctcaagctcttaactgcaacagtggt-3′ (s) and 5′-gtcaaacagaaaattcaaatcccag-3′ (as). In order to obtain the ATM 46-48T Δ mutant a two-step PCR method was used with the following primers 5′-tgagggtacgtatgccctagatg-3′ (s) and 5′-catctagggcatacgtaccctca-3′ (as). From this mutant we obtained ATM 46-48T 41A Δ using primers 5′-cactctactgatgaggatacg-3′ (s) and 5′-cgtatcctcatcagtagagtg-3′ (as). Finally, the primers used to insert the 21–23 substitution in both the ATM Δ and ATM 46-48T Δ mutants were the following: 5′-gtgatataccctcactctac-3′ (s) and 5′-gtagagtgagggtatatcac-3′ (as). On the other hand, to obtain the ATM 5′-new Δ mutant we created a unique StuI site by joining together nucleotides 38–40 (agg) with nucleotides 51–53 (cct). The ATM 5′-new Δ mutation was then introduced by ligating in this site the 5′-gtaggtaagtacgaaggc-3′ (s) and 5′-gccttcgtacttacctac-3′ (as) oligos. Donor splice-site scores were calculated according to the NNSplice 0.9 program available at http://www.fruitfly.org/seq_tools/splice.html (39).Figure 1.

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