Identification of a sequence element directing a protein to nuclear speckles.
SF3b(155) is an essential spliceosomal protein, highly conserved during evolution. It has been identified as a subunit of splicing factor SF3b, which, together with a second multimeric complex termed SF3a, interacts specifically with the 12S U2 snRNP and converts it into the active 17S form. The protein displays a characteristic intranuclear localization. It is diffusely distributed in the nucleoplasm but highly concentrated in defined intranuclear structures termed "speckles," a subnuclear compartment enriched in small ribonucleoprotein particles and various splicing factors. The primary sequence of SF3b(155) suggests a multidomain structure, different from those of other nuclear speckles components. To identify which part of SF3b(155) determines its specific intranuclear localization, we have constructed expression vectors encoding a series of epitope-tagged SF3b(155) deletion mutants as well as chimeric combinations of SF3b(155) sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified (i) a functional nuclear localization signal of the monopartite type (KRKRR, amino acids 196--200) and (ii) a molecular segment with multiple threonine-proline repeats (amino acids 208--513), which is essential and sufficient to confer a specific accumulation in nuclear speckles. This latter sequence element, in particular amino acids 208--440, is required for correct subcellular localization of SF3b(155) and is also sufficient to target a reporter protein to nuclear speckles. Moreover, this "speckle-targeting sequence" transfers the capacity for interaction with other U2 snRNP components.
Eilbracht
J
J
Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Schmidt-Zachmann
M S
MS
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D004252:DNA Mutational Analysis; D006801:Humans; D019913:Nuclear Localization Signals / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; C491799:Sf3b1 protein, mouse; D013912:Threonine; D011392:Proline