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Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.

Kondo Y, Oubridge C, van Roon AM, Nagai K - Elife (2015)

Bottom Line: The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA.The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA.The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

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Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.(A) Filter-binding results for U1 snRNP reconstituted with andwithout U1-C to [32P]-labelled 5′ splice siteoligonucleotide. By curve fitting, the Kd with U1-C is 4.7 ± 0.8 nM andwithout U1-C is 15.8 ± 2.5 nM. CPM, counts per minute. (B)Nucleotides found at each position of the 5′-splice site of theU2-type introns. Adapted from Roca et al.(2008). A, green; C, blue; G, black; U, red. Numbers for highlyconserved positions are highlighted in red. (C) Competitionassays of mutant 5′SS RNA binding to U1 snRNP containing U1C anduncapped but fully modified U1 snRNA. The 5′SS oligonucleotide with+1C, +5C, −1C and +2C substitutions compete weaklywith the wild type oligonucleotide. In panelsC–F, mP is an arbitrary unit offluorescence polarization and error bars indicate standard error.(D) Competition assay with 5′SS oligonucleotides with+3G, +4G, +3U, +4U substitution and the wild type.5′SS oligonucleotide with +2C substitution is included forcomparison. (E) Same as in B except that U1 snRNPlacks U1-C. (F) Same as in C except that U1 snRNPlacks U1-C.DOI:http://dx.doi.org/10.7554/eLife.04986.014
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fig7: Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.(A) Filter-binding results for U1 snRNP reconstituted with andwithout U1-C to [32P]-labelled 5′ splice siteoligonucleotide. By curve fitting, the Kd with U1-C is 4.7 ± 0.8 nM andwithout U1-C is 15.8 ± 2.5 nM. CPM, counts per minute. (B)Nucleotides found at each position of the 5′-splice site of theU2-type introns. Adapted from Roca et al.(2008). A, green; C, blue; G, black; U, red. Numbers for highlyconserved positions are highlighted in red. (C) Competitionassays of mutant 5′SS RNA binding to U1 snRNP containing U1C anduncapped but fully modified U1 snRNA. The 5′SS oligonucleotide with+1C, +5C, −1C and +2C substitutions compete weaklywith the wild type oligonucleotide. In panelsC–F, mP is an arbitrary unit offluorescence polarization and error bars indicate standard error.(D) Competition assay with 5′SS oligonucleotides with+3G, +4G, +3U, +4U substitution and the wild type.5′SS oligonucleotide with +2C substitution is included forcomparison. (E) Same as in B except that U1 snRNPlacks U1-C. (F) Same as in C except that U1 snRNPlacks U1-C.DOI:http://dx.doi.org/10.7554/eLife.04986.014

Mentions: The crystal structure of the minimal U1 snRNP has revealed in detail molecularcontacts between U1 snRNP and a 5′SS RNA with the consensus sequence. The roleof U1-C in stabilising the 5′SS binding was first shown by Heinrichs et al. (1990) using 172 nucleotidepre-mRNA. We measured binding of U1 snRNP to a [32P]-labelled 5′SSoligonucleotide by filter-binding assay (Figure7A). The affinity of U1 snRNP without U1-C (U1 snRNP[ΔU1-C]) for thewild type 5′SS oligonucleotide increases by between three and fourfold onaddition of U1-C (Figure 7A). In order toassess the contribution of the molecular contacts revealed by the crystal structurein 5′SS sequence selection we next assayed binding of variant 5′SSoligonucleotides to U1 snRNP containing uncapped, but otherwise fully authentic, U1snRNA.10.7554/eLife.04986.014Figure 7.Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.


Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.

Kondo Y, Oubridge C, van Roon AM, Nagai K - Elife (2015)

Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.(A) Filter-binding results for U1 snRNP reconstituted with andwithout U1-C to [32P]-labelled 5′ splice siteoligonucleotide. By curve fitting, the Kd with U1-C is 4.7 ± 0.8 nM andwithout U1-C is 15.8 ± 2.5 nM. CPM, counts per minute. (B)Nucleotides found at each position of the 5′-splice site of theU2-type introns. Adapted from Roca et al.(2008). A, green; C, blue; G, black; U, red. Numbers for highlyconserved positions are highlighted in red. (C) Competitionassays of mutant 5′SS RNA binding to U1 snRNP containing U1C anduncapped but fully modified U1 snRNA. The 5′SS oligonucleotide with+1C, +5C, −1C and +2C substitutions compete weaklywith the wild type oligonucleotide. In panelsC–F, mP is an arbitrary unit offluorescence polarization and error bars indicate standard error.(D) Competition assay with 5′SS oligonucleotides with+3G, +4G, +3U, +4U substitution and the wild type.5′SS oligonucleotide with +2C substitution is included forcomparison. (E) Same as in B except that U1 snRNPlacks U1-C. (F) Same as in C except that U1 snRNPlacks U1-C.DOI:http://dx.doi.org/10.7554/eLife.04986.014
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Related In: Results  -  Collection

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fig7: Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.(A) Filter-binding results for U1 snRNP reconstituted with andwithout U1-C to [32P]-labelled 5′ splice siteoligonucleotide. By curve fitting, the Kd with U1-C is 4.7 ± 0.8 nM andwithout U1-C is 15.8 ± 2.5 nM. CPM, counts per minute. (B)Nucleotides found at each position of the 5′-splice site of theU2-type introns. Adapted from Roca et al.(2008). A, green; C, blue; G, black; U, red. Numbers for highlyconserved positions are highlighted in red. (C) Competitionassays of mutant 5′SS RNA binding to U1 snRNP containing U1C anduncapped but fully modified U1 snRNA. The 5′SS oligonucleotide with+1C, +5C, −1C and +2C substitutions compete weaklywith the wild type oligonucleotide. In panelsC–F, mP is an arbitrary unit offluorescence polarization and error bars indicate standard error.(D) Competition assay with 5′SS oligonucleotides with+3G, +4G, +3U, +4U substitution and the wild type.5′SS oligonucleotide with +2C substitution is included forcomparison. (E) Same as in B except that U1 snRNPlacks U1-C. (F) Same as in C except that U1 snRNPlacks U1-C.DOI:http://dx.doi.org/10.7554/eLife.04986.014
Mentions: The crystal structure of the minimal U1 snRNP has revealed in detail molecularcontacts between U1 snRNP and a 5′SS RNA with the consensus sequence. The roleof U1-C in stabilising the 5′SS binding was first shown by Heinrichs et al. (1990) using 172 nucleotidepre-mRNA. We measured binding of U1 snRNP to a [32P]-labelled 5′SSoligonucleotide by filter-binding assay (Figure7A). The affinity of U1 snRNP without U1-C (U1 snRNP[ΔU1-C]) for thewild type 5′SS oligonucleotide increases by between three and fourfold onaddition of U1-C (Figure 7A). In order toassess the contribution of the molecular contacts revealed by the crystal structurein 5′SS sequence selection we next assayed binding of variant 5′SSoligonucleotides to U1 snRNP containing uncapped, but otherwise fully authentic, U1snRNA.10.7554/eLife.04986.014Figure 7.Influence of nucleotide substitutions at the 5′-splice site on U1snRNP binding.

Bottom Line: The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA.The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA.The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

Show MeSH
Related in: MedlinePlus