Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components.
We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a approximately 12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.
Schmidt-Zachmann
M S
MS
Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany.
Knecht
S
S
Krämer
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009928:Organ Specificity; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D013194:Staining and Labeling; D014982:Xenopus laevis
D015415:Biomarkers; D018076:DNA, Complementary; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C088049:splicing factor 3a