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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 nucleotides G132, G133 and U134 in the central hole of the Smprotein assembly.DOI:http://dx.doi.org/10.7554/eLife.04986.009
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fig2s2: The nucleotides G132, G133 and U134 in the central hole of the Smprotein assembly.DOI:http://dx.doi.org/10.7554/eLife.04986.009

Mentions: The structure of the minimal U1 snRNP fits well into the 5.5 Å electron densityexcept for a slight tilt of the duplex between pre-mRNA and the 5′-end of U1snRNA (Figure 1C) (Pomeranz Krummel et al., 2009). In the previous structure thefortuitous interaction between the 5′-end of U1 snRNA from two symmetry-relatedcomplexes could have distorted the orientation of this RNA duplex. Hence we believe thatthe minimal U1 snRNP, with the consensus 5′SS oligonucleotide bound to the5′-end of U1 snRNA, represents the 5′SS-U1 snRNP interaction in the wholeU1 snRNP. The overall structure of the core domain is very similar to that of the U4snRNP core domain, which consists of seven Sm proteins and U4 snRNA (Leung et al., 2011) and the Sm folds of the sevenSm protein assemblies of the two structures (PDB code: 4PJO and 4WZJ) superimpose withrmsd of 0.55 Å. However, there are some noteworthy differences (Figure 2 and Figure 2—figure supplement 1). In the minimal U1 snRNP, helix H0 ofSmD2 points into the minor groove of helix H (Figure1A), buttressing it, a feature already evident at 5.5 Å (Pomeranz Krummel et al., 2009). The nonamer Smsite sequence (AAUUUGUGG in U1 snRNA and AAUUUUUGA in U4 snRNA) has been reported as aminimal RNA oligonucleotide to promote Sm core domain assembly (Raker et al., 1999). In U1 snRNP each base of the first sevennucleotides of the nonamer, AAUUUGU (A125 to U131), interacts one-to-one withSmF-SmE-SmG-SmD3-SmB-SmD1-SmD2 in the pockets formed by four key residues at equivalentpositions in the L3 and L5 loops of the Sm fold except for the interaction of G130 withSmD1 (Figure 2; Figure 2—figure supplement 1). In the protease-treated U1 snRNPstructure (Weber et al., 2010), G132 was placedin the nucleotide-binding pocket in SmF but our structure unambiguously shows that A125occupies this pocket. The U4 core snRNP structure has now been refined to anRfree of 22.4% (Li, Leung, YK, CO and KN, manuscript in preparation; PDBcode: 4WZJ) and the new model shows that whereas previously the G equivalent to G132 wasalso incorrectly placed in SmF (Leung et al.,2011) the Sm site nucleotides bind in a similar manner in both minimal U1 andU4 core domain, except for SmD1. The fourth U of the U4 Sm site sequence is replaced byG in U1 snRNA in the majority of species including humans (http://rfam.sanger.ac.uk/) (Burge et al., 2012). A large guanine base cannot beaccommodated in SmD1 and hence it lies above His37 of SmB outside the central hole(Figure 2; Figure 2—figure supplement 1F). In the Sm site sequence the phosphategroups of A126, U130 and U131 come close and are stabilized by a hydratedMg++ ion (Figure 2). Thelast two nucleotides of the nonamer, G132 and G133, fit into the binding pocket formedby SmD1-SmD2 and SmF-SmE, respectively (Figure2—figure supplement 2).10.7554/eLife.04986.007Figure 2.Stereoview showing binding of U1 snRNA at the central hole of the Smprotein assembly.


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 nucleotides G132, G133 and U134 in the central hole of the Smprotein assembly.DOI:http://dx.doi.org/10.7554/eLife.04986.009
© Copyright Policy
Related In: Results  -  Collection

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

fig2s2: The nucleotides G132, G133 and U134 in the central hole of the Smprotein assembly.DOI:http://dx.doi.org/10.7554/eLife.04986.009
Mentions: The structure of the minimal U1 snRNP fits well into the 5.5 Å electron densityexcept for a slight tilt of the duplex between pre-mRNA and the 5′-end of U1snRNA (Figure 1C) (Pomeranz Krummel et al., 2009). In the previous structure thefortuitous interaction between the 5′-end of U1 snRNA from two symmetry-relatedcomplexes could have distorted the orientation of this RNA duplex. Hence we believe thatthe minimal U1 snRNP, with the consensus 5′SS oligonucleotide bound to the5′-end of U1 snRNA, represents the 5′SS-U1 snRNP interaction in the wholeU1 snRNP. The overall structure of the core domain is very similar to that of the U4snRNP core domain, which consists of seven Sm proteins and U4 snRNA (Leung et al., 2011) and the Sm folds of the sevenSm protein assemblies of the two structures (PDB code: 4PJO and 4WZJ) superimpose withrmsd of 0.55 Å. However, there are some noteworthy differences (Figure 2 and Figure 2—figure supplement 1). In the minimal U1 snRNP, helix H0 ofSmD2 points into the minor groove of helix H (Figure1A), buttressing it, a feature already evident at 5.5 Å (Pomeranz Krummel et al., 2009). The nonamer Smsite sequence (AAUUUGUGG in U1 snRNA and AAUUUUUGA in U4 snRNA) has been reported as aminimal RNA oligonucleotide to promote Sm core domain assembly (Raker et al., 1999). In U1 snRNP each base of the first sevennucleotides of the nonamer, AAUUUGU (A125 to U131), interacts one-to-one withSmF-SmE-SmG-SmD3-SmB-SmD1-SmD2 in the pockets formed by four key residues at equivalentpositions in the L3 and L5 loops of the Sm fold except for the interaction of G130 withSmD1 (Figure 2; Figure 2—figure supplement 1). In the protease-treated U1 snRNPstructure (Weber et al., 2010), G132 was placedin the nucleotide-binding pocket in SmF but our structure unambiguously shows that A125occupies this pocket. The U4 core snRNP structure has now been refined to anRfree of 22.4% (Li, Leung, YK, CO and KN, manuscript in preparation; PDBcode: 4WZJ) and the new model shows that whereas previously the G equivalent to G132 wasalso incorrectly placed in SmF (Leung et al.,2011) the Sm site nucleotides bind in a similar manner in both minimal U1 andU4 core domain, except for SmD1. The fourth U of the U4 Sm site sequence is replaced byG in U1 snRNA in the majority of species including humans (http://rfam.sanger.ac.uk/) (Burge et al., 2012). A large guanine base cannot beaccommodated in SmD1 and hence it lies above His37 of SmB outside the central hole(Figure 2; Figure 2—figure supplement 1F). In the Sm site sequence the phosphategroups of A126, U130 and U131 come close and are stabilized by a hydratedMg++ ion (Figure 2). Thelast two nucleotides of the nonamer, G132 and G133, fit into the binding pocket formedby SmD1-SmD2 and SmF-SmE, respectively (Figure2—figure supplement 2).10.7554/eLife.04986.007Figure 2.Stereoview showing binding of U1 snRNA at the central hole of the Smprotein assembly.

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