Open AccessIdentification of a sequence element directing a protein to nuclear speckles
Author(s) -
Jens Eilbracht,
Marion S. Schmidt-Zachmann
Publication year - 2001
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.071042298
Subject(s) - snrnp , biology , rna splicing , nuclear localization sequence , nuclear export signal , ribonucleoprotein , small nuclear ribonucleoprotein , nucleoplasm , microbiology and biotechnology , spliceosome , cell nucleus , rna , genetics , cytoplasm , gene , nucleolus
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.