Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides.
U7 snRNP is a low-abundance small nuclear ribonucleoprotein particle essential for 3' processing of replication-dependent histone pre-mRNA. We have developed a two-step purification of the particle from TB21 mouse mastocytoma cell nuclear extracts, with about a 20% overall yield, using affinity binding to 2'-O-methyl oligoribonucleotides. The purified particle is homogeneous with respect to RNA content. SDS/PAGE of the U7 snRNP proteins revealed a full complement of the standard core proteins (B, DD', E, F, and G) found in the majority of snRNPs. In addition, two U7-specific polypeptides of 14 kDa and 50 kDa were identified. Summation of the molecular masses of the identified components of the U7 particle yields a particle mass of 249 kDa, in approximate agreement with estimates from sucrose gradient sedimentation (261 kDa) and nondenaturing gradient PAGE (217 kDa).
Smith
H O
HO
Research Institute of Molecular Pathology, Vienna, Austria.
Tabiti
K
K
Schaffner
G
G
Soldati
D
D
Albrecht
U
U
Birnstiel
M L
ML
D000818:Animals; D001483:Base Sequence; D001710:Biotin; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification*; D017411:Ribonucleoproteins, Small Nuclear
D009841:Oligonucleotides; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D001710:Biotin