U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.
The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.
Patton
J R
JR
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Habets
W
W
van Venrooij
W J
WJ
Pederson
T
T
D001323:Autoantibodies; D001483:Base Sequence; D006801:Humans; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000032:analysis; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000378:metabolism* / Q000528:radiation effects; D012261:Ribonucleoproteins / Q000378:metabolism* / Q000528:radiation effects; D017411:Ribonucleoproteins, Small Nuclear; D012685:Sepharose / Q000031:analogs & derivatives; D014466:Ultraviolet Rays; D013205:Staphylococcal Protein A
D001323:Autoantibodies; D008836:Micrococcal Nuclease; D009843:Oligoribonucleotides; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D012685:Sepharose; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A