Informational Suppression to Probe RNA:RNA Interactions in the Context of Ribonucleoproteins: U1 and 5′ Splice-Site Base-Pairing

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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An Intronic Splicing Enhancer Binds U1 snRNPs To Enhance Splicing and Select 5′ Splice Sites

Molecular and Cellular Biology ◽

10.1128/mcb.20.24.9225-9235.2000 ◽

2000 ◽

Vol 20 (24) ◽

pp. 9225-9235 ◽

Cited By ~ 50

Author(s):

Andrew J. McCullough ◽

Susan M. Berget

Keyword(s):

Splice Site ◽

Maximal Activity ◽

Splice Sites ◽

Base Pairing ◽

Rich Region ◽

U1 Snrnp ◽

Splicing Efficiency ◽

Site Recognition ◽

Splicing Enhancer

ABSTRACT Intronic G triplets are frequently located adjacent to 5′ splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5′ splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5′ splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5′ splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.

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Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2960-2965.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2960-2965

Author(s):

E R Suh ◽

R B Waring

Keyword(s):

Splice Site ◽

Site Specificity ◽

Group I Introns ◽

Base Pairing ◽

Group I ◽

Guide Sequence ◽

Compensatory Mutations ◽

Self Splicing ◽

Splice Site Usage ◽

Selection Of

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

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Splice site choice and splicing efficiency are positively influenced by pre-mRNA intramolecular base pairing in yeast

Cell ◽

10.1016/0092-8674(93)90578-e ◽

1993 ◽

Vol 72 (6) ◽

pp. 893-901 ◽

Cited By ~ 41

Author(s):

V GOGUEL

Keyword(s):

Splice Site ◽

Base Pairing ◽

Splicing Efficiency

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Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2960 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2960-2965 ◽

Cited By ~ 29

Author(s):

E R Suh ◽

R B Waring

Keyword(s):

Splice Site ◽

Site Specificity ◽

Group I Introns ◽

Base Pairing ◽

Group I ◽

Guide Sequence ◽

Compensatory Mutations ◽

Self Splicing ◽

Splice Site Usage ◽

Selection Of

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

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