U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C.A wobble pair. Protonation of the C.A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.

Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA. All previous experiments with S P -phosphorothioate-substituted RNAs have been done in the absence of structural information for this particular diastereomer. Yeast U6 RNA contains a metal ion-binding site that is essential for spliceosome function and includes the pro-S P oxygen 5 of U 80 . S P -phosphorothioate substitution at this location creates spliceosomes dependent on thiophilic ions for the first step of splicing. We have determined the solution structure of the U 80 S P -phosphorothioatesubstituted U6 intramolecular stem-loop (ISL), and also report the refined NMR structure of the unmodified U6 ISL. Both structures were determined with inclusion of 1 H- 13C residual dipolar couplings. The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 Å, respectively) allows comparison of the local and long-range structural effect of the modification. We find that the U6-ISL structure is unperturbed by the phosphorothioate. Additionally, the thermodynamic stability of the U6 ISL is dependent on the protonation state of the A 79 -C 67 wobble pair and is not affected by the adjacent phosphorothioate. These results indicate that a single S P -phosphorothioate substitution can be structurally benign, and further validate the metal ion rescue experiments used to identify the essential metal-binding site(s) in the spliceosome.

U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3′ intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67·G80), and disrupts a protonated C67·A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (ΔΔG fold = −3.6 ± 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.Proteomic diversity in eukaryotes is generated by alternative splicing of exons from nuclear premessenger RNA (pre-mRNA) by the spliceosome. The spliceosome is a large ribonucleoprotein complex, made up of five small nuclear RNAs (snRNAs), U1, U2, and U4, U5, U6, and more than 70 proteins (1-4). The spliceosome catalyzes a two-step transesterification reaction, speculated to be RNA-catalyzed by analogy to mitochondrial group II self-splicing ribozymes (2,3,5). Two snRNAs (U2 and U6) likely comprise part of the spliceosome active site, and U6 is essential for catalysis (2,3,5). In the active spliceosome, U2 and U6 form a complex by base pairing to each other, and have also been shown to base pair to pre-mRNA at the first step of splicing. Moreover, mutagenesis data have shown that certain regions of U6 must be intact for the assembled spliceosome to catalyze the first or second step of splicing (6). Other atomic substitution studies of U6 RNA have revealed several phosphate oxygens that are essential for splicing (7-9), which are potential sites of magnesium coordination required by the spliceosome. Recently, the U2-U6 complex was shown to catalyze a reaction similar to the first step of splicing in the † This work was supported by NIH Grant GM65166. S.R.V. absence of protein, lending more evidence to the hypothesis of an RNA active site in the spliceosome (10).The formation of the U2-U6 complex is initiated by a large conformational change in U6 RNA (1, 11). During spliceosome assembly, U6 extensively base pairs with U4 RNA, forming a helical secondary structure. After the spliceosome is assembled completely, base pairing between U4 and U6 is disrupted and U6 undergoes a large conformational change, in which an intramolec...

Summary.  Background: The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. Patients/Methods: We identified a heterozygous 18 base pair, in‐frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer‐intense multimer pattern. This deletion results in loss of amino acids 436–442 of VWFpp, which include one cysteine. Results: Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild‐type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. Conclusions: The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.

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