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Published as doi: 10.1096/fj.07-8377com.
 (The FASEB Journal. 2007;21:3142-3152.)
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Characterization of cyclin L1 as an immobile component of the splicing factor compartment

Andreas Herrmann*,1, Katrin Fleischer{dagger}, Hanna Czajkowska{dagger}, Gerhard Müller-Newen* and Walter Becker{dagger},2

* Institute of Biochemistry and

{dagger} Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Aachen, Germany

2Correspondence: Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany. E-mail: wbecker{at}ukaachen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, A., Fleischer, K., Czajkowska, H., Müller-Newen, G., Becker, W. Characterization of cyclin L1 as an immobile component of the splicing factor compartment.


Key Words: nuclear speckles • photobleaching • arginine-serine-rich domain • mobility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE COMPARTMENTALIZATION OF functions within the nucleus depends on the precise subnuclear targeting of nuclear proteins and subtle control of their mobility. Pre-mRNA splicing factors are known to localize to a specific subnuclear compartment (splicing factor compartment, also referred to as interchromatin granule clusters) that can be visualized by immunofluorescence as a set of irregularly shaped "nuclear speckles" against a diffuse distribution within the nucleoplasm (1) . Although splicing factors are most concentrated in the nuclear speckles, pre-mRNA processing does not take place within these structures (2 , 3) . Therefore, it is thought that the nuclear speckles are sites of assembly, modification, and/or storage of splicing factors from which they shuttle to the sites of active splicing (4) . This view is supported by the observation that speckles increase in size and become round upon inhibition of RNA polymerase II-dependent transcription (e.g., by actinomycin D). These changes presumably reflect the accumulation of unused splicing factors (4) .

Several components of the nuclear speckles have been shown to be highly mobile within the nucleus (5 6 7) , leading to the suggestion that speckles reflect the steady-state association/dissociation of their "resident" proteins within the nucleoplasmic space (4 , 5) . However, time-lapse observations of nuclear speckles in living cells have shown that their positions are largely maintained over many hours (6 , 8) , suggesting that they reside in predetermined locations. So far, it is an open question as to how the nuclear speckles are maintained in a specific territory and whether all components of the speckles exhibit the high mobility within the nucleus (9) .

A defining feature of the nuclear speckles is the presence of SR proteins. Immunofluorescence staining of SC35, a member of the SR family of proteins, is frequently used as a marker of the splicing factor compartment (1 , 10) . Splicing factors of the SR protein family are essential for constitutive splicing and are also important regulators of alternative splicing (11) . SR proteins have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain rich in alternating serine and arginine residues (RS domain) (11) . The RS domain consists of simple repeats of arginine and serine, combined with segments consisting of other amino acids, with an RS content of ~35–40%. RS domains fulfill diverse functions in SR proteins. First, RS domains function as activators of splicing of some, but not all, substrates (12) . Second, the RS domain also serves as a nuclear targeting signal, and nuclear import of SR proteins has been shown to be mediated by a specific member of the importin ß family, transportin-SR, which interacts with the RS domain (13 , 14) . Finally, RS repeats were found to be essential and sufficient for speckle targeting of some SR proteins but not others (15 16 17) .

In addition to the SR family proteins, which contain one or two RRMs and a C-terminal RS domain, there is a heterogeneous group of RS domain-containing proteins (referred to as SR-related proteins) that have variable domain structures (18 , 19) . Most of the SR-related proteins have been implicated in the regulation of chromatin remodeling, transcription, splicing, or mRNA 3' end-processing. Several SR-related proteins have been localized to nuclear speckles by immunofluorescence (18) , suggesting that the RS domain may have a role in subnuclear targeting similar to that in the splicing factors of the SR protein family.

The family of SR-related proteins includes two related proteins, cyclin L1 and cyclin L2, which comprise a N-terminal cyclin domain and a C-terminal RS domain (official gene names, CCNL1 and CCNL2). The sequences of their cyclin domains are 84% identical and are most closely related to those of cyclin K, cyclin T1, and cyclin T2 (30–33% identity). These cyclins form a subfamily of cyclins that are involved in regulating transcription and are clearly separate from the cell cycle-related cyclins (20 , 21) . Cyclin L1 was originally identified as a transcript (designated "ania-6a") that was strongly induced by dopamine or glutamate in striatal neurons (22) . Cyclin L1 and cyclin L2 each exist in at least two different splicing variants, of which the shorter ones contain only an incomplete cyclin domain and lack the RS domain. The long forms of cyclin L1 and L2 appear to be functional cyclins, because they can associate with kinases of the CDK (cyclin-dependent kinase) family and have been shown to stimulate splicing of the ß-globin precursor mRNA in vitro (20 , 23 , 24) . Whereas the short splicing variants of cyclin L1 and L2 are diffusely distributed to the cytoplasm and nucleus, the long forms of cyclins L1 and L2 are colocalized with SC35 in the nuclear speckles (20 , 22) . Surprisingly, we recently found that cyclin L1 and L2 differ strikingly in their intranuclear mobility (20) . Photobleaching experiments revealed that cyclin L2 was highly mobile within the nucleus, as for other RS domain proteins, whereas cyclin L1 was an immobile component of the nuclear speckles.

We have applied fluorescence recovery after photobleaching (FRAP) to further characterize the mobility properties of cyclin L1 and cyclin L2 and to define the structural basis for the retention of cyclin L1 within nuclear speckles. We report that the RS domain of cyclin L1 functions as a nuclear localization signal, directs the protein to nuclear speckles, and is necessary for immobility of cyclin L1 in nuclear speckles of interphase cells. Furthermore, we identified a stretch of basic amino acids whose mutation led to a gain of mobility. Wild-type cyclin L1 was not dislocated from the nuclear speckles after treatment with actinomycin D or overexpression of the protein kinase CLK3. Cyclin L1 becomes mobile during mitosis and is distributed throughout the whole cell in metaphase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression plasmids
The expression clones for GFP fusion proteins of human cyclin L1 and L2 have been described (20) . Only the longest splicing variants containing the RS domain were used in the present study (see supplementary Fig. S1 for the sequences). To produce deletion constructs of GFP-CycL1, silent XbaI sites were introduced at the appropriate sites by site-directed mutagenesis (the recognition sequence for XbaI, TCTAGA, encodes Ser-Arg), and an in-frame stop codon was generated by cutting with XbaI, fill-in, and religation. Site-directed mutagenesis was also used to substitute the KRKK motif for alanine residues. DsRed-CLK3 was constructed by exchanging the GFP-encoding cDNA of pEGFP-CLK3 (20) for the open reading frame encoding DsRed (Discosoma striata red fluorescent protein) from the vector pDsRed-Monomer-N1 (Clontech, Mountain View, CA, USA). Chimera of cyclin L1 and L2 were generated by overlap extension PCR such that the fusion site was localized C-terminal of the cyclin domain within a sequence conserved between both proteins. Specifically, GFP-CycL1/L2 contains amino acids 1–307 of cyclin L1 and 303–521 of cyclin L2, and GFP-CycL2/L1 contains amino acids 1–302 of cyclin L2 and 308–527 of cyclin L1. A construct containing the RS domain of cyclin L1-fused GFP was generated by deleting the sequence coding for amino acids 3–384 from GFP-CycL1. GFP-SF2 contains the entire open reading frame of human SF2 (amino acids 1–248, GenBank accession #M69040) C-terminal of GFP in pEGFP-C1 (Clontech). Sequences of PCR products and mutants were verified by sequencing.

Cell culture and transfections
COS7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal calf serum. Cells were transfected using FuGENE 6 (Roche, Mannheim, Germany), as suggested by the manufacturer. Actinomycin D (Alexis Biochemicals, Lausen, Switzerland) was applied 2 h before analysis at a concentration of 5 µg/ml.

Detection of phosphorylation
COS7 cells were grown in 6-well plates and transfected with 1 µg/well of GFP expression constructs. Two days after transfection, cells were collected in 200 µl ice-cold Nonidet P-40 lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin) and sonicated. The lysates were cleared by centrifugation, and 12 µl aliquots were incubated with or without 15 u alkaline phosphatase (Amersham, Piscataway, NJ, USA) for 30 min at 37°C. Total reactions were then subjected to Western blot analysis, and the expressed proteins were detected with polyclonal rabbit anti-DsRed (1:1000; Clontech) antibody and goat anti-GFP (1:1000; Rockland Inc., Gilbertsville, PA, USA), as indicated in the figure legends. The primary antibodies were labeled with peroxidase-coupled secondary antibodies (AffiniPure rabbit anti-goat IgG from Jackson ImmunoResearch, West Grove, PA, USA; and ImmunoPure goat anti-rabbit IgG from Pierce, Rockford, IL, USA) and detected by chemiluminescence.

Live cell imaging and FRAP analysis
Confocal imaging was carried out on a Zeiss LSM 510Meta confocal microscope equipped with an argon-ion laser and a helium-neon laser (Carl Zeiss, Jena, Germany). Cells transfected with the GFP fusion proteins as indicated were grown on glass coverslips 48 mm in diameter. Forty-eight hours after transfection, living cells were studied at 37°C in DMEM using a temperature-controlled perfusion chamber. The GFP moiety was excited by the 488 nm line of the argon laser and emission was detected using a 505–530 nm band-pass filter. The DsRed moiety of DsRed-CLK3 was excited by the 543 nm line of the helium-neon laser and emission was detected using the Meta-detector adjusted to {lambda} = 563–627 nm. DNA was stained by adding cell-permeable DRAQ5 (Axxora, Lörrach, Germany) to live cells according to the manufacturer’s instructions. Fluorescent DRAQ5 signals were detected as described for DsRed. The images shown here represent confocal slices of ~1 µm. Cells were examined with a 63 x 1.2 NA Zeiss water immersion objective.

FRAP analysis was performed with the same overall setup. After two initial data recordings, bleaching was performed with 100 iterations in a region of interest (ROI). Subsequently, the fluorescence intensity within a ROI was measured every 15 s. Three ROIs were used to calculate normalized fluorescent intensities 1) placed into the bleached region (ROI), 2) surrounding the nucleus, which was bleached (Tot), and 3) outside of the cells to monitor background fluorescence (BG). Normalized fluorescence intensities were calculated from three representative FRAP measurements, as follows (25) :

1) Background subtraction: ROI(t) – BG(t), Tot(t) – BG(t)

2) Correction: (ROI(t) – BG(t))/(Tot(t) – BG(t))

3) Normalization: ((ROI(t) – BG(t))/(Tot(t) – BG(t))) * ((Tot(0) – BG(0))/(ROI(0) – BG(0)))

Immunfluorescence
Detection of GFP fluorescence in paraformaldehyde-fixed cells and detection of endogenous SC35 by indirect immunofluorescence were performed as described (20) . SC35 was detected using the mouse monoclonal anti-SC35 antibody (S4045, Sigma-Aldrich (St. Louis, MO, USA) and either Alexa Fluor 546 (red fluorescence) or Alexa Fluor 488-coupled (green fluorescence) goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) as secondary antibodies. To detect multiple fluorescent signals from one cell, the multitrack function of the LSM 510Meta was used. Fluorescence intensity profiles were generated using the manufacturer’s software (Carl Zeiss, Jena, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FRAP analysis of intranuclear mobility of cyclin L1 and cyclin L2
Cyclin L1 and cyclin L2 are composed of a highly conserved N-terminal cyclin box (84% of identical amino acids) and a more divergent C-terminal RS domain (45% identity; see supplemental Fig. S1 for an alignment). No RNA recognition motif matching the consensus sequences compiled by Maris et al. (26) was identified in either sequence. For live cell imaging experiments, we fused GFP to the N termini of cyclin L1 and cyclin L2 (Fig. 1 A). We have previously shown that GFP fusion proteins of cyclin L1 and cyclin L2 (GFP-CycL1, GFP-CycL2) are colocalized with endogenous SC35 in transiently transfected COS7 cells (20) . In the experiment shown in Fig. 1B , we used FRAP to demonstrate the mobility of GFP-CycL1 and L2 within the nucleus of living COS7 cells. After the bleaching of a strip across the nucleus of cells expressing GFP-CycL2, recovery of fluorescence was nearly complete after 30 s, indicating that the protein was rapidly exchanged for unbleached molecules in the nucleus. In contrast, minimal recovery of fluorescence was detected in GFP-CycL1-transfected cells, indicating that cyclin L1 is immobile within the nucleus.


Figure 1
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  		Figure 1. Mobility of L-type cyclins. A) Domain structure of the fusion proteins GFP-cyclin L1 and GFP-cyclin L2. GFP was fused to the N terminus of cyclin L1 and cyclin L2. The cyclin box and the RS domains are indicated, and the first and last residues of the modules are given. For an alignment of the sequences, see Fig. S1. B) Mobility of cyclin L1 and cyclin L2 as revealed by FRAP analysis. GFP-cyclin L1 or GFP-cyclin L2 were expressed in COS7 cells. Representative interphase nuclei were selected and the GFP moiety was bleached in a region of interest (ROI, white rectangle). Fluorescence recovery after photobleaching was monitored for the time points indicated. Scale bars = 10 µm.

Effect of actinomycin D
Inhibition of RNA polymerase II-dependent transcription leads to characteristic changes in speckle morphology, because the shuttling of splicing factors from the splicing factor compartment to the sites of active transcription is interrupted. Therefore, we asked whether cyclin L1, as an immobile component of the SFC, would be affected by transcriptional arrest. As expected, treatment of COS7 cells with actinomycin D for 2 h caused characteristic changes in the shape and size of nuclear speckles as visualized by SC35 immunofluorescence (Fig. 2 , untransfected). In GFP-CycL2-expressing cells, GFP fluorescence was colocalized with immunolabeled endogenous SC35, as reported before (20) . Upon treatment with actinomycin D, GFP-CycL2 showed the same alterations as SC35, consistent with the hypothesis that cyclin L2 participates in pre-mRNA splicing. In striking contrast, the subnuclear localization of GFP-CycL1 was completely unaffected in actinomycin D-treated cells. Moreover, ectopic expression of GFP-CycL1 prevented the typical changes in localization of SC35 that were observed in untransfected cells. This result indicates that cyclin L1 is not only insensitive to the effects of actinomycin D, but also prevents the effect of actinomycin D on other speckle components.


Figure 2
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  		Figure 2. Sensitivity of L-type cyclins to actinomycin D. COS7 cells were left untransfected or were transfected with expression plasmids for either GFP-cyclin L1 or GFP-cyclin L2 as indicated. The next day, cells were treated with 5 µg/ml actinomycin D (Act. D) for 2 h or left untreated (Ctrl.). After paraformaldehyde fixation, SC35 was immunostained with a specific antibody, and fluorescently labeled secondary antibody. GFP fluorescence (green) and SC35 immunofluorescence (red) were detected by confocal laser scanning microscopy. Scale bars = 10 µm.

Mobility of cyclin L1 during the cell cycle
The speckled pattern of distribution of SR proteins is a feature of interphase cells but disperses upon entry into mitosis (4) . Considering that cyclin L1 is an immobile component of the nuclear speckles, we wanted to study the fate of this protein during mitosis. To identify cells in different phases of the cell cycle, we used the fluorescent live cell dye DRAQ5 to visualize chromatin condensation (27) in a population of asynchronously growing COS7 cells. Maximal compaction of chromatin in metaphase cells was clearly distinguishable from partial condensation in prophase and telophase cells (Fig. 3 A). In addition, nuclei of cells in telophase and in early G1 phase were identified by their pairwise appearance. In prophase cells, before the breakdown of the nuclear envelope, GFP-CycL1 appeared to be concentrated in a reduced number of enlarged subnuclear structures and was excluded from the chromatin. Although this pattern of distribution clearly differed from that observed in interphase nuclei, FRAP analysis revealed that the GFP-CycL1 was still immobile within these structures (Fig. 3B ). Strikingly, in metaphase cells GFP fluorescence was diffusely distributed throughout the cytosol, as observed for other RS domain-containing proteins (28) . FRAP analysis revealed very fast and complete recovery of fluorescence after photobleaching, indicating that GFP-CycL1 was highly mobile in metaphase cells (Fig. 3B ). This result provides clear evidence that the immobility of GFP-CycL1 is a reversible feature and depends on the cellular context. Concomitant with chromatin decondensation in telophase, GFP-CycL1 concentrated again in specific subnuclear structures, where it behaved as an immobile component in FRAP assays. These data show that cyclin L1 underwent changes in its subnuclear distribution similar to those described for mobile components of the nuclear speckles such as SF2 ("speckle cell cycle") (4) .


Figure 3
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  		Figure 3. Distribution and mobility of GFP-cyclin L1 during the cell cycle. For live cell imaging experiments, GFP-cyclin L1 was expressed in COS7 cells. Asynchronously growing cells were incubated with 5 µM DRAQ5 for 20 min to stain DNA. A) Cells representative of the indicated cell cycle phases were identified by their pattern of DRAQ5 fluorescence (red) and monitored by confocal laser scanning microscopy. B) In a live cell imaging experiment, the GFP moiety of GFP-cyclin L1 was bleached in a region of interest (ROI, white circle). Fluorescence recovery after photobleaching (FRAP) was monitored by confocal laser scanning microscopy for the indicated time points. The bleached region is shown in larger magnification in the upper right corner of each picture. Relative fluorescence intensities are shown in false color mode, with highest intensities indicated in red and lowest intensities in blue. Scale bars = 10 µm.

Phosphorylation of the RS domain
Because the mobility of SR proteins is known to be regulated by phosphorylation of the serine residues within the RS domain (29) , we asked whether GFP-CycL1 and GFP-CycL2 were also phosphoproteins. GFP-CycL1 and GFP-CycL2 were expressed in COS7 cells and examined for phosphorylation-dependent mobility shifts in SDS-polyacrylamide gel electrophoresis. To assess whether cyclin L1 and/or cyclin L2 are substrates for CLK kinases, a DsRed-CLK3 fusion protein was cotransfected with L-type cyclins. Members of the CLK family of protein kinases are known to phosphorylate RS domains of SR proteins and can cause the redistribution of speckle proteins (30 , 31) . In addition, we analyzed splicing factor 2 (SF2, also known as alternative splicing factor ASF) as a known substrate of CLK kinases. As shown in the upper right panel of Fig. 4 , a portion of GFP-CycL2 exhibited enhanced electrophoretic mobility after cellular lysates were treated with alkaline phosphatase, indicating that the protein was phosphorylated when expressed in COS7 cells and was partially dephosphorylated by the phosphatase treatment in vitro. Neither higher amounts of phosphatase nor prolonged incubation were able to achieve complete dephosphorylation, suggesting that the phosphorylation sites in cyclin L2 (and cyclin L1) are less susceptible to dephosphorylation than those in SF2 (see below). GFP-CycL1 was detected as a double band (Fig. 4 , upper left panel), and treatment with phosphatase resulted in a slightly increased intensity of the faster migrating band. This result indicates that GFP-CycL1 was partially phosphorylated when expressed in COS7 cells. It is important to note that proteins with RS domains typically exhibit much more pronounced changes in electrophoretic mobility than GFP-CycL1 when dephosphorylated (e.g., refs. 29 , 32 33 34 ; compare SF2 at the bottom right panel of Fig. 4 ), suggesting that cyclin L1 is not extensively phosphorylated in COS7 cells. Because small changes in electrophoretic mobility should be more visible in a protein of lower molecular mass, we created a fusion protein that contained the RS domain of cyclin L1 (residues 385–626) directly fused to the C terminus of GFP (GFP-RScyclin L1). However, electrophoretic mobility of this construct was affected even less by phosphatase treatment (Fig. 4 , bottom left panel), confirming that the RS domain of GFP-CycL1 was not extensively phosphorylated under these conditions. As observed in other cell types (29 , 32 , 35) , GFP-SF2 was detected as a double band in the mock-treated sample and as a single faster migrating band after phosphatase treatment (Fig. 4 , bottom right panel). Coexpression of DsRed-CLK3 increased the intensity of the most slowly migrating form of GFP-SF2, in accordance with earlier results that SF2 is phosphorylated by CLK kinases (30) . For cyclin L1 and cyclin L2, no substantial increase in the intensity of the upper bands was observed. Consistent with this result, overexpression of DsRed-CLK3 in COS7 cells failed to disrupt the punctuate pattern of fluorescence of GFP-CycL1 and GFP-CycL2, whereas SC35 was redistributed from nuclear speckles to diffuse nuclear staining (supplemental material, Fig. S2).


Figure 4
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  		Figure 4. Phosphorylation of the RS domains of cyclin L1 and cyclin L2. GFP-cyclin L1, GFP-cyclin L2, GFP-RScyclin L1, or GFP-ASF/SF2, and either DsRed-CLK3 or the empty vector (Ctrl) were expressed in COS7 cells. Whole-cell lysates were treated with calf intestinal phosphatase (CIP) or were left untreated, as indicated, and separated by SDS-PAGE. Migration of the recombinant proteins was assessed by successive Western blot analysis with antibodies directed against GFP and DsRed.

The mobility properties of cyclin L1 and L2 are defined by regions C-terminal to the cyclin domain
To elucidate whether the cyclin domain of cyclin L1 accounts for the immobility of the protein in nuclear speckles, we constructed domain swap mutants that contained either the cyclin domain of cyclin L1 and the intervening sequence plus the RS domain of cyclin L2, or vice versa. Both chimeric proteins were colocalized with SC35 in nuclear speckles (supplemental Fig. S3). FRAP analysis of single bleached speckles showed that the fluorescence of GFP-CycL1/L2 recovered in a manner similar to that of GFP-CycL2, whereas GFP-CycL2/L1 was immobile (Fig. 5 A). Thus, the region C-terminal of the cyclin box accounts for the immobility of cyclin L1. This result raises the question of whether the RS domain of cyclin L1 alone (amino acids 386–526) is sufficient to keep proteins immobilized in the splicing factor compartment. Therefore, we performed the same analysis with GFP-RScyclin L1. This protein showed the same pattern of distribution within the nucleus as full-length cyclin L1 (supplemental Fig. S3), indicating that the RS domain of cyclin L1 was sufficient for nuclear targeting and for directing the protein to the nuclear speckles. However, FRAP analysis revealed that GFP-RScyclin L1 was highly mobile within the nucleus (Fig. 5B ). This result implies that the capacity of the RS domain of cyclin L1 to immobilize the protein in speckles depends on the presence of more N-terminal parts of the protein (i.e., the cyclin box and/or the intervening sequence between the cyclin box and the first RS repeat) (amino acids 298–384).


Figure 5
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  		Figure 5. Analysis of domain swap constructs of L-type cyclins. A) Wild-type GFP fusion proteins of cyclin L1 and cyclin L2 or chimeric constructs (GFP-cyclin L1/L2, GFP-cyclin L2/L1) were expressed in COS7 cells. The exact fusion sites of the chimera are detailed in Materials and Methods and in supplementary Fig. S1. For FRAP analysis, GFP moieties of the indicated fusion proteins were bleached in a region of interest (ROI, white circle). Fluorescence recovery was monitored by confocal laser scanning microscopy for the time points indicated. The bleached region is shown in larger magnification. Relative fluorescence intensities are shown in false-color mode. B) A deletion mutant of cyclin L1 (GFP-RScyclin L1) was expressed in COS7 cells and subjected to FRAP analysis, as described. Scale bars = 10 µm.

Identification of a sequence necessary for the immobility of cyclin L1 in nuclear speckles
Next we constructed a series of C-terminal deletion mutants of GFP-CycL1 in order to close in on the minimal region of the RS domain required for targeting the protein to and retaining it within the nuclear speckles. The shortest construct (GFP-CycL1{Delta}386) that was truncated at the first RS repeat motif was equally localized in the cytoplasm and the nucleus, and showed no accumulation in subnuclear structures (Fig. 6 A, bottom panels). GFP-CycL1{Delta}426 and the larger constructs were localized in the nucleus, indicating that the sequence region from amino acid 387 to 426 functions as a nuclear targeting sequence (Fig. 6A , upper panels). GFP-CycL1{Delta}426 was already partially colocalized with SC35 in nuclear speckles, but also exhibited diffuse nucleoplasmic staining. Subnuclear localization of GFP-CycL1{Delta}464 and GFP-CycL1{Delta}472 was indistinguishable from that of the full-length protein.


Figure 6
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  		Figure 6. Analysis of C-terminal deletion mutants of cyclin L1. GFP-cyclin L1 and the indicated C-terminal deletion constructs were expressed in COS7 cells. A) Subcellular localization of the GFP fusion proteins and colocalization with SC35 were analyzed by confocal laser scanning microscopy of SC35-immunostained cells. B) For FRAP analysis, the GFP moieties of the indicated fusion proteins were bleached in a region of interest (ROI, white circle), and fluorescence recovery was observed by confocal laser scanning microscopy for the time points indicated. Scale bars = 10 µm. C) In a quantitative FRAP experiment, three measurements of fluorescence recovery after photobleaching of each GFP fusion protein were evaluated. The means of corrected and normalized fluorescence intensities were calculated and depicted as a function of time.

Next we performed FRAP experiments in order to determine whether the deletion constructs were mobile components of the splicing factor compartment. After bleaching of single nuclear speckles, GFP-CycL1{Delta}472 showed no detectable recovery of fluorescence within 60 s, indicating that the protein was immobile in the speckles and behaved in a manner identical to that of full-length GFP-CycL1 (Fig. 6B ). In contrast, a considerable recovery of GFP fluorescence was observed after the bleaching of cells transfected with GFP-CycL1{Delta}426, indicating that this deletion construct was at least partially mobile. GFP-CycL1{Delta}464 showed an intermediate behavior in that it showed detectable recovery of fluorescence within 60 s, but appeared to be less mobile than GFP-CycL1{Delta}426. To more precisely determine the different mobilities of the deletion constructs, we performed quantitative FRAP analysis. Figure 6C shows changes of the corrected and normalized fluorescence intensities (means of three cells) over time. This evaluation confirmed that wild-type GFP-CycL1 and GFP-CycL1{Delta}472 were immobile components of the nuclear speckles, whereas GFP-CycL1{Delta}464 showed partial recovery of fluorescence, and GFP-CycL1{Delta}426 was highly mobile.

These FRAP results indicate that the segment of the RS domain between amino acids 427 and 472 accounts for the difference between a mobile (GFP-CycL1{Delta}426) and an immobile construct of cyclin L1 (GFP-CycL1{Delta}472), and that amino acids 464–472 are required for maximal immobilization in nuclear speckles. We hypothesized that the sequence responsible for retaining cyclin L1 within nuclear speckles, if physiologically relevant, should be evolutionarily conserved. Guided by sequence comparisons of cyclin L1 orthologs from different vertebrates (Fig. 7 A) and by the different mobilities of GFP-CycL1{Delta}464 and GFP-CycL1{Delta}472, we wondered whether a cluster of positively charged amino acids between 466 and 479 might be important for the mobility properties of cyclin L1. To test this idea, we constructed a point mutant of full-length GFP-CycL1 in which the residues K466R467K468K469 were replaced by alanines. This mutant protein was colocalized with SC35 in nuclear speckles (data not shown). FRAP analysis showed partial recovery of fluorescence in bleached speckles (Fig. 7B ). This result indicates that the mutated residues are essential for maximal immobility of cyclin L1 in the nuclear speckles.


Figure 7
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  		Figure 7. A basic sequence motif within the RS domain of cyclin L1 is essential for complete immobility within nuclear speckles. A) Sequence alignment of cyclin L1 from different vertebrates (upper panel). Only the region harboring the putative speckle retention signal is shown. The C-terminal amino acid of each deletion construct ({Delta}426, {Delta}464, {Delta}472) and the amino acids exchanged for alanines (AAAA) by site-directed mutagenesis are indicated. Only amino acids differing from human cyclin L1 are shown. RS dipeptide motifs are highlighted in blue. Two short segments of 6 or 12 nonaligned amino acids were omitted (angle brackets). B) FRAP analysis of GFP-cyclin L1mutKRKK was performed as described in Fig. 6 . The graph represents the quantitative evaluation of fluorescence recovery (means of measurements from three cells) over time. Scale bars = 10 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The application of live cell microscopy combined with advanced bleaching techniques has revealed that many proteins, although clearly concentrated in specific subnuclear structures, actually move in and out of these structures very rapidly (5 , 36 , 37) . These proteins include components of the nuclear speckles such as the SR proteins SF2 and SC35, which are extremely dynamic both within and outside speckles (5 , 6 , 8 , 38) . In this study we have characterized the SR-related protein cyclin L1 as the first known example of a protein that is an immobile component of nuclear speckles and show that a segment of the RS domain (amino acids 427–472) is required for speckle retention.

The present results add to the increasing evidence that RS domains of individual SR proteins and SR-related proteins are not functionally equivalent but account for differences in subcellular targeting and intracellular mobility. RS domains function as nuclear localization signals in most if not all SR proteins, but differ in their ability to direct splicing factors to nuclear speckles. The RS domains of two Drosophila splicing regulators, SWAP and Tra (15 , 16) , and the mammalian SR proteins SRp20 and SC35 are known to direct these splicing factors to nuclear speckles, whereas the RS domain of SF2/ASF is neither necessary nor sufficient for speckle localization (17) . Cyclin L1 and cyclin L2 are also localized in nuclear speckles, and here we show that the RS domain, and more specifically a segment of 39 amino acids (387–426; containing 12 RS dipeptides), is sufficient for nuclear targeting and for the subnuclear pattern of distribution of cyclin L1. It should be noted that the localization of a protein in nuclear speckles is not defined by targeting signals because there are neither gates nor directed transport within the nucleus. Instead, localization within speckles is thought to be a consequence of its steady-state accumulation by transient interactions with locally immobilized binding sites (39) . According to this model, the apparent immobility of cyclin L1 in nuclear speckles could be explained as the consequence of a particularly tight interaction with an unknown binding partner. Alternatively, it is conceivable that cyclin L1 is a component of the elusive structural scaffold of the speckles. However, the highly inducible expression of cyclin L1 argues against this possibility (22) .

The domain swap experiments show that the striking difference in mobility between cyclin L1 and L2 is determined by the C-terminal part of the protein (amino acids 308–526). However, the RS domain of cyclin L1 alone was highly mobile within the nucleus, indicating that the immobility in the nuclear speckles depends on the presence of further structural elements (i.e., the cyclin domain or the intervening region between the cyclin domain and the RS domain). Analysis of the deletion constructs identified a short region within the RS domain (amino acids 427–472) that was necessary for retention of GFP-cyclin L1 in the speckle compartment (Fig. 6B, C ). This segment showed no sequence similarity with cyclin L2 (see supplementary Fig. S1) but was evolutionarily conserved in the cyclin L1 orthologs from chicken, frog (Xenopus), and fish (Fig. 7A ). Within this region, we found a conserved cluster of four basic residues (K466R467K468K469) to be essential for the maximal degree of immobility of cyclin L1, but not for localization of the protein in nuclear speckles. However, GFP-CycL1mKRKK was less mobile than GFP-CycL1{Delta}426, suggesting that the protein-protein interaction that presumably accounts for speckle retention was not completely disrupted by this mutation. In conclusion, these results show that the difference in mobility between cyclin L1 and cyclin L2 is due to the specific sequences of the RS domains, and provide evidence of the functional heterogeneity of RS domains in different proteins. Similarly, a specific sequence within the RS domain of SC35 has been identified that acts as a nuclear retention signal and prevents nucleocytoplasmic shuttling of this SR protein (32) . However, in contrast to cyclin L1, SC35 is highly mobile within the nucleus (38) .

The intranuclear mobility of SR proteins is regulated by phosphorylation of serine residues within the RS domain (29) . In particular, overexpression of members of the CLK family of protein kinases causes the redistribution of all speckle proteins tested to a diffuse nuclear localization, leaving no residual structure, as revealed by electron microscopy (9 , 30 , 31) . It was therefore unexpected that both cyclin L1 and cyclin L2 were still found in speckle-like structures when coexpressed with CLK3 (supplementary Fig. S2), whereas SC35 was completely dislocated from nuclear speckles. Partial resistance of the L-type cyclins toward the effect of CLK3 corresponds with our observation that their phosphorylation was only marginally enhanced, if at all, by overexpression of CLK3 (Fig. 4) . Notably, the results of phosphatase treatment revealed an interesting difference between cyclin L1 and cyclin L2: the mobile speckle protein cyclin L2 was clearly phosphorylated in COS7 cells, possibly by a kinase with a different substrate specificity than CLK3. Kinases that can phosphorylate RS domains include SR protein kinases 1 and 2 (SRPK1 and SRPK2) (40 , 41) . In contrast to cyclin L2, electrophoretic mobility of cyclin L1 was hardly altered by phosphatase treatment, providing strong evidence that the RS domain of cyclin L1 was not extensively phosphorylated. Because serine phosphorylation of the RS domain is known to be required for the recruitment of SR proteins, from nuclear speckles to transcription sites in vivo (29) , it is noticeable that the lack of phosphorylation of cyclin L1 correlates with its immobility in the speckles. However, this experiment does not exclude the possibility that the lack of phosphorylation is a consequence rather than a cause of the immobility of cyclin L1.

A trivial explanation for the immobility and lack of phosphorylation of GFP-cyclin L1 would be that the overexpressed fusion protein artificially forms insoluble aggregates within the nucleus. This appears very unlikely because of, among other reasons, the graded effects of the C-terminal deletions and the specific effect of the point mutation. Moreover, our observation that cyclin L1 undergoes the normal speckle cell cycle (4) , becoming diffusely distributed throughout metaphase cells, provides clear evidence that cyclin L1 is not irreversibly immobilized in the speckles. Changes in the pattern of distribution of cyclin L1 were also evident in prophase and telophase, when the protein was still (or already) immobile. Thus, the immobility of cyclin L1 in interphase nuclei does not preclude the typical changes of speckle morphology in the cell cycle. So far, it is not clear exactly how the disintegration and reassembly of the speckles in the course of the cell cycle are regulated, and it remains to be determined how cyclin L1 becomes mobile in metaphase and immobile again in telophase.

In the presence of active gene expression, splicing factors are thought to constantly shuttle from nuclear speckles to the sites of splicing catalysis, and reverse. Inhibition of gene expression disrupts the recruitment of splicing factors to active genes and thus increases the steady-state content of these factors in the speckles. The resulting changes in speckle morphology were evident for cyclin L2, whereas cyclin L1 showed no change in its subnuclear distribution upon treatment of the cells with actinomycin D (Fig. 2) . This result is consistent with the expectation that immobile components of the speckles should not be affected by inhibition of transcription, and suggests that cyclin L1 is not directly involved in the regulation of transcription or gene expression. However, it is possible that the protein can be mobilized under certain circumstances. The lack of an effect of actinomycin D appears to contradict earlier results that cyclin L1 acts as a splicing factor (23) . Recently, overexpression of cyclin L1 was shown to favor selection of the most distal alternative splice site of the adenovirus-derived E1A minigene (42) . This is the opposite effect of that obtained by overexpression of the alternative splicing factors SC35 or SF2, and was explained by an inhibitory effect of cyclin L1/CDK12 on SC35 and SF2. Because CDK12 is also known to be localized in SC35 speckles (43) , cyclin L1 does not need to be mobile to counteract the effect of other SR proteins on alternative splicing. Strikingly, in our experiments, overexpression of GFP-cyclin L1 prevented the effects of actinomycin D on SC35 localization (Fig. 2C ), suggesting that cyclin L1 may cause the retention of splicing factors in the speckles and thereby antagonize their effects on splicing. It is important to note that cyclin L1 (in contrast to cyclin L2) is a highly inducible protein with low basal expression (22) . One might speculate that the induction of cyclin L1 is a mechanism to regulate alternative splicing by preventing other RS domain proteins from leaving the splicing factor compartment.


   ACKNOWLEDGMENTS
 
We thank Oliver Spelten for initial FRAP analyses of the domain swap chimera. This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants to W.B. (Be-1967/2–1) and G.M.N. (SFB542 TP B12 and Z1).


   FOOTNOTES
 
1 Present address: Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, CA 91010, USA.

Received for publication March 9, 2007. Accepted for publication April 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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