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Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors.

Culler SJ, Hoff KG, Voelker RB, Berglund JA, Smolke CD - Nucleic Acids Res. (2010)

Bottom Line: Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance.In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE.Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

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Enriched ISRE hexamers demonstrate silencer activity. (A) Mutational analysis of ISS5 supports the silencing activity of individual hexamer regions. The combined and individual activity of hexamer regions within the context of ISS5 was examined by introducing 2 point mutations into 3 hexamer regions in combination and separately. Sequences were characterized in transient transfection assays in HEK-293 cells. For all reported data, silencing activity was assessed by flow cytometry analysis, where the mean GFP levels from two independent experiments were normalized to the NMD control. Normalized expression and average error are reported. P-values derived from the Student’s t-test are as follows: *P < 0.05 and **P < 0.01. (B) Mutational analysis of ISS8 supports the silencing activity of combined hexamer regions. The combined and individual activity of hexamer regions within the context of ISS8 was examined by introducing 2 point mutations into 2 hexamer regions in combination and separately. (C) Individual hexamer analysis supports the silencing activity of enriched hexamer sequences. Hexamers and corresponding mutant and duplicate sequences were characterized in transient transfection assays in HEK-293 cells. The mean GFP levels from two independent experiments were normalized to the wild-type hexamer construct.
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Figure 4: Enriched ISRE hexamers demonstrate silencer activity. (A) Mutational analysis of ISS5 supports the silencing activity of individual hexamer regions. The combined and individual activity of hexamer regions within the context of ISS5 was examined by introducing 2 point mutations into 3 hexamer regions in combination and separately. Sequences were characterized in transient transfection assays in HEK-293 cells. For all reported data, silencing activity was assessed by flow cytometry analysis, where the mean GFP levels from two independent experiments were normalized to the NMD control. Normalized expression and average error are reported. P-values derived from the Student’s t-test are as follows: *P < 0.05 and **P < 0.01. (B) Mutational analysis of ISS8 supports the silencing activity of combined hexamer regions. The combined and individual activity of hexamer regions within the context of ISS8 was examined by introducing 2 point mutations into 2 hexamer regions in combination and separately. (C) Individual hexamer analysis supports the silencing activity of enriched hexamer sequences. Hexamers and corresponding mutant and duplicate sequences were characterized in transient transfection assays in HEK-293 cells. The mean GFP levels from two independent experiments were normalized to the wild-type hexamer construct.

Mentions: Most of the recovered ISRE sequences contain several enriched n-mers, where 88% of all extended 15-mers (plus 2-nt flanking region) contain at least one enriched hexamer which comprise the largest group of n-mers in our data set (Supplementary Table S11). ISS5 contains seven enriched hexamers resembling binding sites for the CELF and SF2/ASF proteins, which cluster into three main regions within the sequence (Figure 4A). We introduced two point mutations within each region and in combination and assessed their activity through transient transfection assays. Mutations within each region decrease expression to levels comparable to the NMD control, supporting that each region has regulatory activity. Simultaneous mutations to regions 2 and 3 resulted in expression levels comparable to or slightly higher than the individual mutations, indicating that the regulatory function of ISS5 is likely not due to combinatorial recognition of motifs.Figure 4.


Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors.

Culler SJ, Hoff KG, Voelker RB, Berglund JA, Smolke CD - Nucleic Acids Res. (2010)

Enriched ISRE hexamers demonstrate silencer activity. (A) Mutational analysis of ISS5 supports the silencing activity of individual hexamer regions. The combined and individual activity of hexamer regions within the context of ISS5 was examined by introducing 2 point mutations into 3 hexamer regions in combination and separately. Sequences were characterized in transient transfection assays in HEK-293 cells. For all reported data, silencing activity was assessed by flow cytometry analysis, where the mean GFP levels from two independent experiments were normalized to the NMD control. Normalized expression and average error are reported. P-values derived from the Student’s t-test are as follows: *P < 0.05 and **P < 0.01. (B) Mutational analysis of ISS8 supports the silencing activity of combined hexamer regions. The combined and individual activity of hexamer regions within the context of ISS8 was examined by introducing 2 point mutations into 2 hexamer regions in combination and separately. (C) Individual hexamer analysis supports the silencing activity of enriched hexamer sequences. Hexamers and corresponding mutant and duplicate sequences were characterized in transient transfection assays in HEK-293 cells. The mean GFP levels from two independent experiments were normalized to the wild-type hexamer construct.
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Related In: Results  -  Collection

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Figure 4: Enriched ISRE hexamers demonstrate silencer activity. (A) Mutational analysis of ISS5 supports the silencing activity of individual hexamer regions. The combined and individual activity of hexamer regions within the context of ISS5 was examined by introducing 2 point mutations into 3 hexamer regions in combination and separately. Sequences were characterized in transient transfection assays in HEK-293 cells. For all reported data, silencing activity was assessed by flow cytometry analysis, where the mean GFP levels from two independent experiments were normalized to the NMD control. Normalized expression and average error are reported. P-values derived from the Student’s t-test are as follows: *P < 0.05 and **P < 0.01. (B) Mutational analysis of ISS8 supports the silencing activity of combined hexamer regions. The combined and individual activity of hexamer regions within the context of ISS8 was examined by introducing 2 point mutations into 2 hexamer regions in combination and separately. (C) Individual hexamer analysis supports the silencing activity of enriched hexamer sequences. Hexamers and corresponding mutant and duplicate sequences were characterized in transient transfection assays in HEK-293 cells. The mean GFP levels from two independent experiments were normalized to the wild-type hexamer construct.
Mentions: Most of the recovered ISRE sequences contain several enriched n-mers, where 88% of all extended 15-mers (plus 2-nt flanking region) contain at least one enriched hexamer which comprise the largest group of n-mers in our data set (Supplementary Table S11). ISS5 contains seven enriched hexamers resembling binding sites for the CELF and SF2/ASF proteins, which cluster into three main regions within the sequence (Figure 4A). We introduced two point mutations within each region and in combination and assessed their activity through transient transfection assays. Mutations within each region decrease expression to levels comparable to the NMD control, supporting that each region has regulatory activity. Simultaneous mutations to regions 2 and 3 resulted in expression levels comparable to or slightly higher than the individual mutations, indicating that the regulatory function of ISS5 is likely not due to combinatorial recognition of motifs.Figure 4.

Bottom Line: Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance.In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE.Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

Show MeSH