The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU 4-6 GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2 hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU. . .) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.Small nuclear ribonucleoproteins (snRNPs) mediate essential RNA processing events, including pre-mRNA splicing (reviewed in references 43 and 45). The major spliceosomal snRNP particles consist of snRNA (U1, U2, U4/U6, or U5) and numerous proteins, which can be classified as either snRNP specific or common to each snRNP particle. The common proteins are collectively referred to as Sm proteins and were originally identified due to their antigenicity in the autoimmune disease systemic lupus erythematosus (26). The best characterized of these are the seven HeLa Sm proteins, which are named B/BЈ, D1, D2, D3, E, F, and G and range in size from 241 (for BЈ) to 76 (for G) amino acids (reference 16 and references therein). B and BЈ are alternatively spliced products of the same gene, differing only in 11 amino acids at their C termini (42), and are referred to here as B/BЈ.Association of the Sm proteins with U snRNA plays a pivotal role in the further biogenesis of U snRNP particles. The metabolic stability of the U snRNP particles is dependent on the Sm protein association (10,20,37). Following export from the nucleus, each U snRNA (with the exception of U6) assembles cytoplasmically with a set of Sm proteins, resulting in its core RNP particle. Hypermethylation of the U snRNA 5Ј cap structure from monomethyl guanosine (m 7 G) to trimethylguanosine (m 3 G) is dependent upon formation of the core RNP domain, and in particular requires the presence of the B/BЈ and D3 Sm proteins (30,34,36). These Sm proteins are the last to assemble (36, 38), linking cap hypermethylation to th...