THE ASSOCIATION OF THE U1-SPECIFIC 70K AND C-PROTEINS WITH U1 SNRNPS IS MEDIATED IN PART BY COMMON U-SNRNP PROTEINS

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K a nd A proteins are known to bind directly to stem-loops of the U1 snRNA , whereas the U1-C protein does not bind to naked U1 snRNA, but depend s on other U1 snRNP protein components for its association. Focusing o n the U1-70K and U1-C proteins, protein-protein interactions contribut ing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after inc ubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revea led that both common snRNP proteins and the U1-70K protein are require d for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70 K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did no t bind naked U1 RNA, associated stably with core U1 snRNPs. This sugge sts that a new U1-70K binding site is generated upon association of co mmon U1 snRNP proteins with U1 RNA. The interaction between the N-term inal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, sugg esting essential structural differences among snRNP core domains. Evid ence for direct protein-protein interactions between U1-specific prote ins and common snRNP proteins was supported by chemical crosslinking e xperiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presente d in which the complex of common proteins on the RNA backbone function s as a platform for the association of the U1-specific proteins.