Identification of a sequence element directing a protein to nuclear speckles

Abstract

SF3b¹⁵⁵ 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 SF3b¹⁵⁵ suggests a multidomain structure, different from those of other nuclear speckles components. To identify which part of SF3b¹⁵⁵ determines its specific intranuclear localization, we have constructed expression vectors encoding a series of epitope-tagged SF3b¹⁵⁵ deletion mutants as well as chimeric combinations of SF3b¹⁵⁵ 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 SF3b¹⁵⁵ 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.