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 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of 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. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in 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 between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.
Hartmuth
K
K
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mankopff-Strasse 2, Marburg, D-35037, Germany.
Raker
V A
VA
Huber
J
J
Branlant
C
C
Lührmann
R
R
D000241:Adenosine; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D001665:Binding Sites; D002467:Cell Nucleus; D008745:Methylation; D009690:Nucleic Acid Conformation; D009865:Oocytes; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D014529:Uridine; D014982:Xenopus laevis; D055517:snRNP Core Proteins
D001324:Autoantigens; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; D000241:Adenosine; D014529:Uridine