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Previous Article | Next Article 
Molecular and Cellular Biology, May 2006, p. 3468-3477, Vol. 26, No. 9
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.9.3468-3477.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607
Received 23 December 2005/ Returned for modification 2 February 2006/ Accepted 6 February 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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E2Fs are very loosely classified into two groups according to their function. E2F1, E2F2, and E2F3a are generally considered activators, whereas E2F3b, E2F4, and E2F5 have the properties of repressors. Accordingly, the combined loss of E2f-1, E2f-2, and E2f-3 in primary mouse fibroblasts results in the repression of E2F-dependent transcription and a block in cell proliferation (50). Recent microarray and chromatin immunoprecipitation studies have led to the appreciation that besides regulation of G1-to-S progression, E2F/pRB has many other diverse cellular functions, including regulation of G2/M progression, apoptosis, DNA repair, DNA recombination, and differentiation (16). There is also considerable debate over the mechanism by which pRB negatively controls cell proliferation and its tumor suppressor properties. Thus, a large number of related E2F and pRB family members in mammalian cells and recent findings that these proteins have a role beyond the G1/S control complicate study of the function and regulation of these proteins in vivo.
Drosophila melanogaster has a streamlined version of the mammalian cell cycle regulation. This is attested by the functional conservation of the basic biochemical machinery that controls cell proliferation in flies and mammals (12). In flies, there are two pRB-related genes, rbf1 and rbf2, and two E2F genes, an activator, dE2F1, and a repressor, dE2F2 (11, 17, 20, 22, 37, 42, 45). The two dE2Fs work antagonistically during development. Consistent with the function of E2F, de2f1 mutant flies are retarded in growth and show clear defects in S-phase entry and in the G1/S transcriptional program (19, 40). The loss of the repressor de2f2 does not significantly impact development and has a rather minimal effect on E2F-dependent transcription. Strikingly, many features of the de2f1 mutant phenotype, including the block to cell proliferation, are rescued by concomitant loss of de2f2 (22). There are several ways to explain this result, and at present, it is not clear precisely how de2f1 and de2f2 antagonize each other. Nevertheless, the de2f1 mutant phenotype is evidently a result of unchecked de2f2 activity and modulation of de2f2 function is able to rescue the de2f1 mutant phenotype.
There has been significant progress in recent years toward understanding of the biochemical properties of the E2F and pRB proteins and the ways they are regulated. Nevertheless, there is no agreement about the significance of these features in vivo. Apparently, our understanding of the roles of E2F and pRB and their regulation in the context of a multicellular organism lags far behind the pace of their biochemical characterization. We are particularly interested in identification of new genes that interface with the E2F/pRB module in vivo and contribute to its regulation during development. In this work, we show that the loss of a splicing factor, SR protein B52, overcomes the dE2F2-dependent block to cell proliferation in de2f1 mutant cells. In B52-deficient cells, the level of dE2F2 protein is severely reduced and endogenous dE2F2 target genes become derepressed. We show that these defects correlate with incorrect splicing of the de2f2 pre-mRNA. To our knowledge, this is the first example in which a mutation in a gene other than de2f2 is capable of rescuing the de2f1 mutant phenotype in vivo. Thus, our data establish a functional significance of B52 regulation of dE2F2 and establish a mechanistic link between splicing and cell cycle progression.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The FRT82B B525d21 de2f1+ chromosome was generated from the FRT82B B525d21 de2f1729 chromosome by precise excision of the P element from de2f1 gene in the de2f1729 allele.
To select B525d21 homozygous mutant larvae for Western blot analysis, the B52 mutant chromosome was balanced over the TM6B balancer containing the actin-green fluorescent protein (Act-GFP) transgene. The homozygous mutants were identified by the absence of a GFP signal under a dissecting microscope.
Manipulations with SL2 cells. RNA interference (RNAi) in SL2 cells was carried out using double-stranded RNA (dsRNA) as described previously (44), except that cells were retreated with dsRNA after 2 days. T7-tailed primers specific for targeted RNAs were designed to amplify an 800-bp fragment. The PCR product was then used to synthesize an RNA with the Ribomax-T7 RNA transcription kit (Promega). After precipitation, two strands of RNA were annealed overnight into dsRNA. The efficiency of protein depletion was monitored by Western blot analysis.
For transfections, the Flag-tagged B52 open reading frame was cloned into vector pIE4 (Novagen) under the control of the baculovirus ie1 promoter, the construct was verified by sequencing, and the expression of the protein was verified by Western blot analysis using a mouse anti-Flag antibody (Sigma). The E2F reporter construct p5'-168DPCNAluc, the ß-galactosidase-expressing construct pIE4-lacZ, the dE2F1-expressing construct pIE-dE2F1, and the dE2F2-expressing construct pIE-dE2F2 were described previously (22, 42).
Transient transfections in Drosophila SL2 cells were performed using CellFectin reagent (Invitrogen) according to the manufacturer's recommendations with 10 µg of plasmid DNA. For Western blot analysis, 5 x 106 to 10 x 106 cells were lysed in 250 µl of 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, and 0.25% NP-40, pH 7.8, buffer and frozen for 5 min at –80°C. After spinning, the protein concentration was determined by the Bradford assay (Bio-Rad). Proteins were resolved by electrophoresis on sodium dodecyl sulfate-10% polyacrylamide. The following antibodies were used for a subsequent Western blot analysis: rabbit polyclonal anti-B52 (13), DX-3 mouse monoclonal anti-RBF1 (17), Yun-3 anti-dDP (18), guinea pig polyclonal anti-dE2F1 (7), and rabbit polyclonal anti-dE2F2 (22).
For quantitative real-time PCR, RNA was isolated from SL2 cells using Trizol and treated for 1 hour at 37°C with RNase-free DNase RQ1 (Promega). The RQ1 was then removed by purification with the RNAeasy kit (QIAGEN); 1 µg of total RNA was used to synthesize a cDNA with the iScript cDNA synthesis kit (Bio-Rad). One-fiftieth of the cDNA synthesis reaction and primer sets was mixed with 2x iQ SYBR green supermix (Bio-Rad). Real-time PCR was performed using a MyIQ PCR machine (Bio-Rad). Primers for real-time PCR were designed using Beacon Designer 4.0 software. To control for the presence of genomic DNA in the reaction, the control PCR was carried out using equivalent amounts of RNA instead of cDNA as a template. Several repeats of the reverse transcription (RT)-PCR assays using multiple RNA preparation of independent RNAi treatments produced the same results.
The following primers were used for real-time PCR: de2f1, TCGCCTTTGTGTCCATCAATCC and CATAGGCATCCGAACCGAAGTC; de2f2, ACTATCACCGTCTGGACAC and AAGTTTCATCTCATCTTTCATCC; dDP, GGACACGGATGCGGATGG and GTGCGGCTCCTGACTAACC; Arp53D, CCTTGGCATAGTTCATTGACACATAGCAG and GGGCGTTACAATGGGCACCTCT; PCNA, GCCGGTGACGCTGACATTTG and TACTCGACTACCAGCGGAACATCT; ß-tubulin, ACATCCCGCCCCGTGGTC and AGAAAGCCTTGCGCCTGAACATAG; and CG17142, AGGGTCCAGCTCTGCGTCATC and CAACCCCAATATCGTGCCCATCA.
The following primers were used for RT-PCR to detect splicing products of the de2f2 mRNA: primer pair A, CACGTGATGATCTGCTGGACATC and AGGATTCAGCTGCAGCAGC, and primer pair B, CTCGTGAAGGCCAACGAAGG and TCGTATATTCGGCGCTTCTGTACG.
Immunofluorescence. Bromodeoxyuridine labeling of eye imaginal discs was performed as previously described using DNase instead of HCl to preserve the GFP signal (46). For immunostaining, mouse monoclonal anti-cyclin B (Developmental Studies Hybridoma Bank) and anti-phosphorylated histone H3 (Upstate) antibodies were used. Images were collected on a Zeiss LSM510 confocal microscope.
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RESULTS |
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We sequenced a B52 open reading frame from the B525d21 mutant chromosome and found that it contains a point mutation that converts a starting AUG into AUA. In agreement with this, no B52 protein is detected by Western blot analysis in extracts prepared from whole B525d21 mutant larvae (Fig. 1C). To unambiguously prove that the B52 mutation is responsible for the suppression, we found that a known B52 allele, B52s2249, also rescues the de2f1 mutant phenotype (data not shown). Taken together, these results strongly argue that the B52 gene encodes a genuine suppressor of the de2f1 mutant phenotype.
Next, we examined clones of B525d21 de2f1729 double mutant cells in the eye imaginal discs dissected from third-instar larvae. Mutant tissue was distinguished from wild-type tissue by the absence of GFP signal (Fig. 1D). As expected, clones of de2f1729 mutant cells are tiny and the eye disc consists entirely of wild-type cells (Fig. 1E). However, numerous clones of B525d21 de2f1729 double mutant cells are clearly recognizable in the eye disc (Fig. 1F).
We compared the eyes bearing clones of B525d21 de2f1729 (Fig. 1F) double mutant cells to clones of dDP (Fig. 1G) mutant cells. dDP is a heterodimeric partner of E2F and removing dDP impairs E2F function as efficiently as targeting dE2F1 and dE2F2 (23). Thus, clones of dDP mutant cells are essentially identical to clones of de2f1 de2f2 double mutant cells. Since the overall amount of B525d21 de2f1729 mutant tissue roughly equals the amount of dDP mutant tissue (compare Fig. 1F and G), this indicates that the loss of B52 might have a strong impact on dE2F2 function. Although this cannot be used as an accurate measurement of cell proliferation, it gives a rough estimation of the strength of a suppressor mutation.
We generated clones of B52 mutant cells on the background of the wild-type de2f1 allele. Our concern was that the loss of B52 may cause tissue overgrowth and "compensate" for the loss of dE2F1 instead of affecting the function of the dE2F2 repressor complex. Following clone induction, we did not find any evidence of overrepresentation of the B525d21 de2f1+ homozygous mutant tissue compared to wild-type tissue (Fig. 1H). A similar result was obtained using the B52s2249 mutant allele (data not shown). We inferred that the rescue by B52 mutation of the de2f1 mutant phenotype is not due to tissue overgrowth.
To further characterize B52 de2f1 double mutant cells, the pattern of S phases was visualized by bromodeoxyuridine labeling of the eye discs following clone induction. The cell cycles in the eye imaginal discs is developmentally regulated. In wild-type cells of the imaginal disc, the morphogenetic furrow separates the cells asynchronously cycling in the anterior part of the disc from the cells synchronously entering the last S phase that is called the second mitotic wave (Fig. 2A). Such synchrony is achieved by imposing a G1 arrest in the morphogenetic furrow. After the second mitotic wave, cells permanently exit the cell cycle and commit to a differentiation program. S phases occur normally in clones of the B525d21 de2f1729 double mutant (Fig. 2A and B) and B525d21 single mutant cells (Fig. 2D and E). The double mutant cells show correct timing for entering and exiting the second mitotic wave. Importantly, no additional bromodeoxyuridine labeling occurs after the second mitotic wave or within the morphogenetic furrow. Thus, the loss of B52 does not interfere with the onset of differentiation, permanent cell cycle exit, or cell quiescence. It appears that the loss of B52 is capable of relieving the S-phase block in de2f1 mutant cells exactly when the cells are normally scheduled to initiate replication.
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B52 is required for dE2F2-mediated repression. Since multiple B52 mutant alleles restore proliferation in de2f1-deficient cells, we concluded that B52 is a genuine modifier of the de2f1 mutant phenotype. To gain insights into the mechanism of the B52 rescue, we employed RNA interference in Drosophila SL2 tissue culture cells. Cells were incubated with B52 double-stranded RNA, and the level of B52 protein was determined by Western blot analysis. As a control, cells were treated in parallel with nonspecific dsRNA. Following 4 days of treatment, the level of B52 protein was severely reduced (Fig. 3A). Thus, RNAi is efficient in depleting cells of B52 protein.
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The level of Arp53D and CG17142 in B52-depleted cells was quantified using a real-time PCR assay. ß-Tubulin mRNA was used as a standard since its level is not affected by depletion of either dE2Fs or B52 (15, 28). The plotted graphs represent the ratios of the abundances of the corresponding transcripts in cells treated with specific dsRNAs compared to that of control treated cells. In cells treated with dE2F2 dsRNA, Arp53D and CG17142 are strongly derepressed (Fig. 3B). Depletion of B52 resulted in the derepression of both Arp53D and CG17142 to extents similar to those seen in cells depleted of dE2F2. In contrast to group E genes, depletion of dE2F2 does not have an effect on the expression of a well-known E2F target, PCNA (Fig. 3C). In a similar way, PCNA transcripts are not deregulated in B52-treated cells. Thus, depletion of B52 in essence phenocopies the effects on E2F-dependent transcription observed in dE2F2-deficient cells.
Having established that the expression of endogenous dE2F2-specific targets is dependent on B52, we asked whether overexpression of B52 has an effect on the expression of a PCNA reporter construct in transient-transfection assays. This reporter is generally used as a readout of the dE2F2 activity (22, 24, 42, 44, 47). Flag-tagged full-length B52 cDNA was cloned into the expression vector under the control of the baculovirus promoter ie1. An increasing amount of B52 expression construct was cotransfected with the E2F reporter into SL2 cells and the level of exogenous B52 expression was followed using anti-Flag antibody (data not shown). The lacZ expression plasmid was used to normalize for transfection efficiency. B52 strongly represses the E2F reporter, and this effect can be titrated by increasing amounts of B52 expression plasmid (Fig. 4A).
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Depletion of B52 affects dE2F2 protein and mRNA levels. Having established that B52 requires dE2F2 to represses an E2F reporter, we asked whether B52 affects the dE2F2 level. Cells were treated with B52 dsRNA and the level of dE2F2 was monitored by Western blot analysis after 1, 2, and 4 days of incubation. As shown in Fig. 5A, B52 is efficiently depleted 1 day after treatment and the B52 protein does not reappear on day 2 or 4. Strikingly, the dE2F2 level is dramatically decreased in B52-depleted cells on day 1 and remains barely detectable on days 2 and 4. This effect is specific to dE2F2 because the dE2F1 level is not affected in B52-deficient cells (Fig. 5B). Since the B52 DNA sequence does not share any homology with dE2F2, this effect is unlikely to be due to a nonspecific targeting of dE2F2 by B52 dsRNA. To further exclude the possibility of nonspecific effects of B52 dsRNA on dE2F2 protein, we used two nonoverlapping fragments of B52 cDNA to produce dsRNA probes: one corresponds to the 5' region, B52 (1 to 400), and the other corresponds to the 3' region, B52 (400 to 800). As shown in Fig. 5C, both dsRNAs, as well as a dsRNA synthesized from a larger fragment of B52 (1 to 800), were equally efficient in depletion of B52 protein, and in each case the dE2F2 protein level was severely reduced. Importantly, similar effects were also observed in mutant animals. Early larval lethality associated with the loss of B52 efficiently precluded us from using imaginal discs of mutant animals for Western blot analysis. Instead, we chose the larval brain as a source of material since B52 was shown to be essential for splicing in this tissue (27) and the brain can be straightforwardly dissected even at early stages of larval development. Protein extracts were prepared from larval brains of early second-instar larvae of wild-type and B525d21 de2f1729 double mutant flies. In agreement with the RNAi experiments, dE2F2 protein was dramatically reduced in the mutant animals (Fig. 5D). Thus, the loss of B52 results in reduction of dE2F2 both in cells and in animals.
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To determine whether the changes in dE2F2 and dDP protein levels are due to transcriptional effects, we quantified de2f2 and dDP mRNAs in B52-depleted cells by real-time PCR. No decrease in the levels of dDP and de2f1 transcripts was observed in B52-depleted cells (Fig. 6). However, de2f2 mRNA was severely reduced in B52-deficient cells as evidenced by the real-time PCR assay using a pair of primers specific for exon 4. From these experiments, we concluded that the loss of B52 leads to a reduction in the level of de2f2 mRNA and dE2F2 protein. Since dDP transcripts appear to be unaffected in B52-depleted cells and the level of dDP protein has been shown to be reduced in the absence of dE2F2 protein (23), the reduced dDP level is most likely to be a consequence of a low level of dE2F2 protein.
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Two sets of primers, A and B, were chosen for RT-PCR assays to amplify segments containing de2f2 exon boundaries (Fig. 7A). No RT was used to control for the presence of genomic DNA in the RNA sample. Splicing product B between exon 1 and 2 in de2f2 pre-mRNA is detected in both control treated and B52-deficient cells (Fig. 7B). However, de2f2 mRNA is not correctly spliced between exons 3 and 5 in B52-depleted cells. This product was not detected either in a multiplex PCR with both A and B sets of primers or in a PCR assay using only the primers A (Fig. 7B). This is consistent with the 15-fold reduction in the level of properly spliced de2f2 mRNA in B52-depleted cells when a pair of primers specific for exon 4 was used (Fig. 6). Since the wild-type splicing product between exons 1 and 2 is present in B52-depleted cells at an approximately normal level, we concluded that the absence of B52 affects the splicing of the de2f2 pre-mRNA rather than its stability or transcription. A reduction in properly spliced mRNA level was previously observed for several other B52 targets (6, 28).
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To distinguish between the two possibilities, we transfected de2f2 cDNA into cells depleted of B52 by RNAi or into cells treated with a nonspecific dsRNA. The ability of dE2F2 to repress was determined by measuring expression from the cotransfected E2F reporter. As shown in Fig. 8, dE2F2 represses the reporter in B52-depleted cells and the level of repression is indistinguishable from that observed in control treated cells. These data indicate that defective splicing of de2f2 mRNA is most likely to be the primary cause of the loss of E2F-mediated repression in B52-deficient cells.
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DISCUSSION |
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Numerous studies on pRB and E2F have led to the identification of a large number of proteins that were proposed to be essential for pRB-induced cell cycle arrest. However, in most cases very little is known about their contribution to pRB function in vivo. Here, we used a genetic approach in Drosophila and identified the B52 gene, which is required for dE2F2/RBF to block cell proliferation in de2f1 mutant cells. Using three different B52 mutant alleles, we found that the size of clones of de2f1 B52 double mutant cells is dramatically increased in comparison to that of clones of de2f1 mutant cells.
There are two possible explanations for the increased clone size of de2f1 mutant cells in the absence of B52. One possibility is that clones of de2f1 B52 double mutant cells are defective in their ability to permanently withdraw from the cell cycle. This seems unlikely since we do not find any evidence of ectopic S phases or mitoses in B52 de2f1 double mutant cells in the eye disc. On the contrary, the patterns of cell proliferation appear to be strikingly normal in mutant cells, indicating that the mutant cells respond properly to physiological proliferative and antiproliferative signals. We further note that the timing of S phases is indistinguishable between adjacent wild-type and de2f1 B52 double mutant cells. Thus, we favor the explanation that de2f1 mutant cells progress through the G1/S block at a relatively normal rate in the absence of B52.
Interestingly, de2f1 B52 double mutant cells show mild defects in G2/M progression. This perhaps explains why clones of de2f1 B52 double mutant cells are smaller than clones generated from the wild-type parental chromosome. The phenotype of de2f1 B52 double mutant cells is strikingly similar to the phenotype of dDP mutant cells. Since the loss of dDP mimics simultaneous inactivation of de2f1 and de2f2 (23), this may indicate that de2f2 activity is severely compromised in B52-deficient cells. Such an interpretation is further supported by a strong reduction of the dE2F2 protein level in mutant animals and in cells depleted of B52 by RNAi. The reduction in protein level is accompanied by derepression of dE2F2/RBF target genes, suggesting that dE2F2 function is indeed efficiently abrogated by inactivation of B52.
We noted that de2f1 B52 double mutants die at the first- and second-instar larval stages. This is likely to be a consequence of the early larval lethality associated with the loss of B52 (39), although we cannot exclude the possibility that the B52 mutation does not rescue the de2f1 mutant phenotype in all cell types. It has previously been shown that B52 makes a different contribution to the splicing of endogenous targets in various tissues (27).
Although cell cycle progression and splicing are, at first glance, two independent processes, there is a growing body of evidence suggesting an intimate link between them. This link is best exemplified in Saccharomyces cerevisiae, in which a number of genes that were isolated in genetic screens for splicing factors (prp screens) were independently identified in cell cycle screens (cdc screens) and vice versa (2-4, 14, 41). It is unlikely that the cell cycle arrest is simply a consequence of global defects in splicing since mutations in most genes encoding splicing factors do not perturb the cell cycle. On the contrary, a limited number of mutations simultaneously affect splicing and cell cycle progression. For example, mutations in the CDC40/PRP17 gene, which encodes a pre-mRNA splicing factor involved in the second step of the splicing reaction (49), result in G2/M arrest and defects in G1-to-S progression (14). Similarly, the CDC5/Cef1 product was shown to be a component of the spliceosome that is directly implicated in splicing (1). cdc5 mutants arrest during the G2 phase of the cell cycle prior to entry into mitosis (36). These examples are not limited to yeast, since the loss of SR protein ASF/SF2 in a chicken B-cell line results in G2-phase cell cycle arrest and programmed cell death (30).
In spite of the wealth of data, the exact molecular mechanisms underlying the link between the cell cycle and splicing remain elusive. It has been suggested that specific splicing factors may be selectively required for efficient splicing of genes that directly or indirectly regulate progression through the cell cycle. This is supported by several studies in which the cell cycle defects were linked to abnormal splicing of a particular gene. For example, in cdc40 mutants, the ANC1 gene appear to be a critical target (14), although the precise role of ANC1 in cell cycle control is unclear. G2/M arrest caused by mutation in the splicing factor CDC5/Cef1 is due to inefficient splicing of an intron in the 
-tubulin gene (9). Nevertheless, these studies failed to directly link a splicing factor to a known cell cycle regulator. Furthermore, although the loss of ASF/SF2 brings about G2 arrest, no significant differences in the level of any transcripts encoding known cell cycle regulators were found (29). It was suggested that the observed cell cycle arrest is likely due to activation of a DNA damage checkpoint induced by the appearance of double-strand DNA breaks upon the loss of ASF/SF2 (30).
Our data provide support for the idea of a direct connection between pre-mRNA splicing and the cell cycle. The genetic and molecular data presented here implicate the well-characterized SR protein B52 in the specific regulation of splicing of the de2f2 pre-mRNA that encodes a core component of the cell cycle machinery. B52 is a member of the serine/arginine-rich (SR) protein family of essential splicing regulators. SR proteins contain one or two conserved RNA recognition motifs and a variable arginine/serine-rich (RS) domain. SR proteins are thought to participate in several steps of the splicing reaction and function as both general and regulatory splicing factors. Their regulatory roles are attributed to diverse RNA binding specificities by RNA recognition motifs (25, 26).
A large body of work firmly establishes that B52 is a splicing factor. In vitro, B52 complements both mammalian and Drosophila splicing-deficient cytoplasmic S100 extracts (27, 33). In mutant animals, B52 is not required for global RNA processing but is rate limiting for the splicing of a subset of pre-mRNAs in certain tissues (27). In agreement with this, we find that splicing of de2f2 pre-mRNA but not de2f1 or dDP pre-mRNA is affected in B52-deficient cells. Thus, the role of B52 in splicing of the de2f2 pre-mRNA appears to be highly specific. Genetic rescue of the de2f1 mutant phenotype by mutation in the B52 gene further underscores the functional requirement for B52 in the dE2F2/RBF-dependent cell cycle block in vivo.
Recent microarray experiments identified approximately 100 alternatively spliced pre-mRNAs that are aberrantly regulated in B52-deficient tissue culture cells (6). We cannot exclude the possibility that deregulation of targets other than de2f2 in B52-depleted cells may also affect dE2F2-mediated repression. Our data do not support such an interpretation, however, since reexpression of the intronless de2f2 cDNA that is insensitive to regulation by B52 is sufficient to restore dE2F2-mediated repression in B52-deficient cells. Thus, we suggest that abnormal splicing of the de2f2 pre-mRNA is likely to be a primary cause of the loss of dE2F2-mediated repression.
Further insights into specific regulation of the de2f2 pre-mRNA splicing by B52 will likely require identification of signaling pathways that may utilize B52 to modulate the level of processed de2f2 mRNA. This may provide the cell with an alternative way to effectively inactivate the dE2F2/RBF complex in response to certain signals. It is worth noting that the cdk2-cyclin E complex was shown to phosphorylate the U2 snRNP protein SAP155 (43), exemplifying how the activity of the spliceosome can be modulated in response to the position of the cell within the cell cycle.
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ACKNOWLEDGMENTS |
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This work was supported in part by American Cancer Society Illinois Division grant 05-26 to M.V.F. M.V.F. is a Leukemia & Lymphoma Society Special Fellow.
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FOOTNOTES |
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