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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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The U1 snRNA constructs used for this work.(A) Full-length U1 snRNA is used for in vitro reconstitutionand further biochemical experiments. Sequences within dashed rectanglesindicate the 5′-end variations of U1 snRNA used in biochemicalexperiments. U1 + Ψ construct contains both 2′-O-methylgroups on A1 and U2 as well as pseudo-uridines at position 5 and 6. U1− Ψ construct has 2′-O-methyl modifications, but hasnormal uridines instead of pseudo-uridines. (B) The minimal U1snRNA (SmKCm) construct replaces the 4-way junction with a kissing loop.(C) In SL1·SL2 RNA, the apical region of stem-loop 2(U1 snRNA nucleotides 61–78) is shown in red, stem-loop 1 (U1 snRNAnucleotides 17–47) in black and other nucleotides are shown in cyan.Bases in stem-loop 1 and 2 are numbered as in natural human U1 snRNA.DOI:http://dx.doi.org/10.7554/eLife.04986.005
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fig1s2: The U1 snRNA constructs used for this work.(A) Full-length U1 snRNA is used for in vitro reconstitutionand further biochemical experiments. Sequences within dashed rectanglesindicate the 5′-end variations of U1 snRNA used in biochemicalexperiments. U1 + Ψ construct contains both 2′-O-methylgroups on A1 and U2 as well as pseudo-uridines at position 5 and 6. U1− Ψ construct has 2′-O-methyl modifications, but hasnormal uridines instead of pseudo-uridines. (B) The minimal U1snRNA (SmKCm) construct replaces the 4-way junction with a kissing loop.(C) In SL1·SL2 RNA, the apical region of stem-loop 2(U1 snRNA nucleotides 61–78) is shown in red, stem-loop 1 (U1 snRNAnucleotides 17–47) in black and other nucleotides are shown in cyan.Bases in stem-loop 1 and 2 are numbered as in natural human U1 snRNA.DOI:http://dx.doi.org/10.7554/eLife.04986.005

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)

The U1 snRNA constructs used for this work.(A) Full-length U1 snRNA is used for in vitro reconstitutionand further biochemical experiments. Sequences within dashed rectanglesindicate the 5′-end variations of U1 snRNA used in biochemicalexperiments. U1 + Ψ construct contains both 2′-O-methylgroups on A1 and U2 as well as pseudo-uridines at position 5 and 6. U1− Ψ construct has 2′-O-methyl modifications, but hasnormal uridines instead of pseudo-uridines. (B) The minimal U1snRNA (SmKCm) construct replaces the 4-way junction with a kissing loop.(C) In SL1·SL2 RNA, the apical region of stem-loop 2(U1 snRNA nucleotides 61–78) is shown in red, stem-loop 1 (U1 snRNAnucleotides 17–47) in black and other nucleotides are shown in cyan.Bases in stem-loop 1 and 2 are numbered as in natural human U1 snRNA.DOI:http://dx.doi.org/10.7554/eLife.04986.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383343&req=5

fig1s2: The U1 snRNA constructs used for this work.(A) Full-length U1 snRNA is used for in vitro reconstitutionand further biochemical experiments. Sequences within dashed rectanglesindicate the 5′-end variations of U1 snRNA used in biochemicalexperiments. U1 + Ψ construct contains both 2′-O-methylgroups on A1 and U2 as well as pseudo-uridines at position 5 and 6. U1− Ψ construct has 2′-O-methyl modifications, but hasnormal uridines instead of pseudo-uridines. (B) The minimal U1snRNA (SmKCm) construct replaces the 4-way junction with a kissing loop.(C) In SL1·SL2 RNA, the apical region of stem-loop 2(U1 snRNA nucleotides 61–78) is shown in red, stem-loop 1 (U1 snRNAnucleotides 17–47) in black and other nucleotides are shown in cyan.Bases in stem-loop 1 and 2 are numbered as in natural human U1 snRNA.DOI:http://dx.doi.org/10.7554/eLife.04986.005
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