Characterization of cyclin L1 as an immobile component of the splicing factor compartment.
Cyclin L1 and cyclin L2 are two closely related members of the cyclin family that contain C-terminal arginine- and serine-rich (RS) domains and are localized in the splicing factor compartment (nuclear speckles). Here we applied photobleaching techniques to show that a green fluorescent protein (GFP) fusion protein of cyclin L1, in contrast to cyclin L2, was not mobile within the nucleus of living COS7 cells. The objectives of this study were to 1) characterize the intranuclear localization and mobility properties of cyclin L1 in different cellular states, and 2) dissect the structural elements required for immobilization of cyclin L1. Transcriptional arrest by actinomycin D caused accumulation of GFP-cyclin L2 in rounded and enlarged nuclear speckles but did not affect the subnuclear pattern of distribution of GFP-cyclin L1. Although immobile in most phases of the cell cycle, GFP-cyclin L1 was diffusely distributed and highly mobile in the cytoplasm of metaphase cells. By analysis of a series of chimeras, deletion constructs, and a point mutant, a segment within the RS domain of cyclin L1 was identified to be necessary for the immobility of the protein in nuclear speckles. This study provides the first characterization of an immobile component of nuclear speckles.
Herrmann
Andreas
A
Institute of Biochemistry, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
Fleischer
Katrin
K
Czajkowska
Hanna
H
Müller-Newen
Gerhard
G
Becker
Walter
W
D000595:Amino Acid Sequence; D000818:Animals; D000880:Anthraquinones / Q000378:metabolism; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D003609:Dactinomycin / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones; C468873:CCNL1 protein, human; C483080:CCNL2 protein, human; D016213:Cyclins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003609:Dactinomycin