Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA.
Nucleotide analog interference mapping (NAIM) is a powerful method for identifying RNA functional groups involved in protein-RNA interactions. We examined particles assembled on modified U1 small nuclear RNAs (snRNAs) in vitro and detected two categories of interferences. The first class affects the stability of two higher-order complexes and comprises changes in two adenosines, A65 and A70, in the loop region previously identified as the binding site for the U1 small nuclear ribonucleoprotein (snRNP)-specific U1A protein. Addition of an exocyclic amine to position 2 of A65 interferes strongly with protein binding, whereas removal or modification of the exocyclic amine at position 6 makes little difference. Modifications of A70 exhibit the opposite effects: Additions at position 2 are permitted, but modification of the exocyclic amine at position 6 significantly inhibits protein binding. These interactions, critical for U1A-U1 snRNA recognition in the context of in vitro snRNP assembly, are consistent with previous structural studies of the isolated protein with the RNA hairpin containing the U1A binding site. The second category of interferences affects all partially assembled U1-protein complexes by decreasing the stability of Sm core protein associations. Interestingly, most strong interferences occur at phosphates in the terminal stem-loop region of U1, rather than in the Sm binding site. These data argue that interactions with the phosphate backbone of the terminal stem loop are essential for the stable association of Sm core proteins with the U1 snRNA. We suggest that the stem loop of all Sm snRNAs may act as a clamp to hold the ring of Sm proteins in place.
McConnell
Timothy S
TS
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, CT 06536, USA.
Lokken
R Peter
RP
Steitz
Joan A
JA
D006801:Humans; D011485:Protein Binding / Q000502:physiology; D012342:RNA, Small Nuclear / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000096:biosynthesis* / Q000378:metabolism
D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA