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.
Highlights
IntroductionRemoval of introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression
Removal of introns from pre-messenger RNA is an essential step in eukaryotic gene expression
A minimal U1 small nuclear ribonucleoprotein particles (snRNPs) 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)
Summary
Removal of introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression. This process is catalysed by a large and dynamic RNA-protein assembly called the spliceosome, which consists of five small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs) and numerous non-snRNP proteins (Will and Lührmann, 2011). U1 snRNP recognizes a short sequence at the 5′-splice site (5′SS) of pre-mRNA through basepairing between the 5′-end of U1 snRNA and the 5′SS sequence (Lerner et al, 1980; Zhuang and Weiner, 1986; Siliciano and Guthrie, 1988; Séraphin et al, 1988) and promotes an ordered assembly of the four remaining snRNPs to form the spliceosome, which undergoes extensive conformational and compositional remodelings to become catalytically active (Will and Lührmann, 2011). The most striking feature of the structure is the N-terminal region of U1-70k, which extends from its RRM through a long α-helix and wraps around the Sm protein assembly
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