Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.
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.
Raker
V A
VA
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, 35037 Marburg, Germany.
Hartmuth
K
K
Kastner
B
B
Lührmann
R
R
D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009843:Oligoribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism* / Q000648:ultrastructure; D017381:Spliceosomes / Q000378:metabolism*
D009843:Oligoribonucleotides; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear