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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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Protein constructs used in this study.(A) The schematic diagram of the domain architecture of U1snRNP proteins is shown with horizontal bars. The thinner lines below thebars represent the constructs used for crystallization: blue lines forsequences used in the U1A70kF fusion protein, red lines for sequences usedin the 70kSmD1F fusion protein, and black lines indicate the extent ofprotein constructs used in minimal U1 crystallisation. For binding assays,U1 snRNP was reconstituted with full-length proteins except U1-70k(2–216) and SmB (1–174). (B) The fusion proteinconstructs were designed using a (Gly–Ser) linker (GS, pink). U1A70kFis used for the determination of U1-70k (60–216) fragment withStem-loop I RNA. 70kSmD1F is used for the minimal U1 snRNP domaincrystallization.DOI:http://dx.doi.org/10.7554/eLife.04986.004
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fig1s1: Protein constructs used in this study.(A) The schematic diagram of the domain architecture of U1snRNP proteins is shown with horizontal bars. The thinner lines below thebars represent the constructs used for crystallization: blue lines forsequences used in the U1A70kF fusion protein, red lines for sequences usedin the 70kSmD1F fusion protein, and black lines indicate the extent ofprotein constructs used in minimal U1 crystallisation. For binding assays,U1 snRNP was reconstituted with full-length proteins except U1-70k(2–216) and SmB (1–174). (B) The fusion proteinconstructs were designed using a (Gly–Ser) linker (GS, pink). U1A70kFis used for the determination of U1-70k (60–216) fragment withStem-loop I RNA. 70kSmD1F is used for the minimal U1 snRNP domaincrystallization.DOI:http://dx.doi.org/10.7554/eLife.04986.004

Mentions: A minimal U1 snRNP consisting of seven Sm proteins, the N-terminal peptide of U1-70k,U1-C and a truncated U1 snRNA was designed based on the 5.5 Å resolution structure(Figure 1A; Figure 1—figure supplements 1 and 2) (Pomeranz Krummel et al., 2009). A large portion ofRNA attached to Helix H through the 4-way junction was replaced by a kissing-loop (Ennifar et al., 2001) to facilitate crystalcontacts (Figure 1—figure supplement 2).However, this eliminated the binding site (stem-loop I) for the U1-70k RRM(RNA-recognition motif) (Query et al., 1989)and thus weakened the binding of the U1-70k N-terminal peptide to the snRNP core domain(Nelissen et al., 1994; Hilleren et al., 1995). In order to stabilize itsbinding we fused the N-terminal 59 residue peptide of U1-70k to SmD1 via aGly–Ser linker (70kSmD1F, Figure1—figure supplement 1). The reconstituted complex was stable andyielded crystals diffracting to 3.3 Å (Table1). The second crystal (U1A70kF-RNA) contains residues 60–216 of U1-70kand the entire U1 snRNA stem-loop I. In order to promote interaction of the longα-helix of U1-70k with the RNA stem we fused it to the U1-A RRM and capped the RNAwith the apical loop of stem-loop II such that the α-helix is anchored to the RNAstem (Figure 1—figure supplements 1 and2). The crystal structure of this complex was determined at 2.5 Å (Figure 1B; Table1). Phases for both crystal structures were determined by molecular replacement.10.7554/eLife.04986.003Figure 1.Crystal structures of the two sub-structures of U1 snRNP.


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)

Protein constructs used in this study.(A) The schematic diagram of the domain architecture of U1snRNP proteins is shown with horizontal bars. The thinner lines below thebars represent the constructs used for crystallization: blue lines forsequences used in the U1A70kF fusion protein, red lines for sequences usedin the 70kSmD1F fusion protein, and black lines indicate the extent ofprotein constructs used in minimal U1 crystallisation. For binding assays,U1 snRNP was reconstituted with full-length proteins except U1-70k(2–216) and SmB (1–174). (B) The fusion proteinconstructs were designed using a (Gly–Ser) linker (GS, pink). U1A70kFis used for the determination of U1-70k (60–216) fragment withStem-loop I RNA. 70kSmD1F is used for the minimal U1 snRNP domaincrystallization.DOI:http://dx.doi.org/10.7554/eLife.04986.004
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383343&req=5

fig1s1: Protein constructs used in this study.(A) The schematic diagram of the domain architecture of U1snRNP proteins is shown with horizontal bars. The thinner lines below thebars represent the constructs used for crystallization: blue lines forsequences used in the U1A70kF fusion protein, red lines for sequences usedin the 70kSmD1F fusion protein, and black lines indicate the extent ofprotein constructs used in minimal U1 crystallisation. For binding assays,U1 snRNP was reconstituted with full-length proteins except U1-70k(2–216) and SmB (1–174). (B) The fusion proteinconstructs were designed using a (Gly–Ser) linker (GS, pink). U1A70kFis used for the determination of U1-70k (60–216) fragment withStem-loop I RNA. 70kSmD1F is used for the minimal U1 snRNP domaincrystallization.DOI:http://dx.doi.org/10.7554/eLife.04986.004
Mentions: A minimal U1 snRNP consisting of seven Sm proteins, the N-terminal peptide of U1-70k,U1-C and a truncated U1 snRNA was designed based on the 5.5 Å resolution structure(Figure 1A; Figure 1—figure supplements 1 and 2) (Pomeranz Krummel et al., 2009). A large portion ofRNA attached to Helix H through the 4-way junction was replaced by a kissing-loop (Ennifar et al., 2001) to facilitate crystalcontacts (Figure 1—figure supplement 2).However, this eliminated the binding site (stem-loop I) for the U1-70k RRM(RNA-recognition motif) (Query et al., 1989)and thus weakened the binding of the U1-70k N-terminal peptide to the snRNP core domain(Nelissen et al., 1994; Hilleren et al., 1995). In order to stabilize itsbinding we fused the N-terminal 59 residue peptide of U1-70k to SmD1 via aGly–Ser linker (70kSmD1F, Figure1—figure supplement 1). The reconstituted complex was stable andyielded crystals diffracting to 3.3 Å (Table1). The second crystal (U1A70kF-RNA) contains residues 60–216 of U1-70kand the entire U1 snRNA stem-loop I. In order to promote interaction of the longα-helix of U1-70k with the RNA stem we fused it to the U1-A RRM and capped the RNAwith the apical loop of stem-loop II such that the α-helix is anchored to the RNAstem (Figure 1—figure supplements 1 and2). The crystal structure of this complex was determined at 2.5 Å (Figure 1B; Table1). Phases for both crystal structures were determined by molecular replacement.10.7554/eLife.04986.003Figure 1.Crystal structures of the two sub-structures of U1 snRNP.

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