An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 co ntain a common set of eight Sm proteins that bind to the conserved single-s tranded 5'-PuAU(3-6)GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vit ro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting o f in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. Ln contrast wit h the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as w ell as an adenosine two nucleotides downstream of the Sm binding site, show ed an unexpected reactivity with dimethyl sulfate. This novel reactivity co uld be attributed to N7-methylation of the adenosine and was not observed i n naked RNA, indicating that it is an intrinsic property of the RNA-protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found bet ween the unusual N7-A methylation sensitivity of the Sm core RNP and its ab ility to be imported into the nucleus of Xenopus laevis oocytes. (C) 1999 A cademic Press.