![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 10, 4187-4199, October 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal; Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, MA 02129; and Departamento de Ciências da Saúde, Universidade da Madeira, 9000-390 Funchal, Portugal
Submitted January 13, 2006;
Revised July 10, 2006;
Accepted July 11, 2006
Monitoring Editor: Karsten Weis
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF35a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF35a or U2AF35b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF35a is essential for HeLa cell division and suggest a novel role for both U2AF35 protein isoforms as regulators of alternative splicing of a specific subset of genes. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
The initial events of spliceosome assembly require recognition of specific sequences at the 5' and 3' splice junctions, as well as other sequences in the pre-mRNA. In both yeast and mammals, the 5' splice site is initially recognized by U1 snRNP, whereas the 3' end of introns in higher eukaryotes is recognized by the U2 auxiliary factor (U2AF). U2AF is a heterodimer composed of a large (U2AF65) and a small (U2AF35) subunit. U2AF65 binds to the pyrimidine-rich (Py) tract upstream of the 3' splice junction (Zamore et al., 1992), and U2AF35 binds to the AG dinucleotide at the 3' splice site (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999a).
In contrast to U2AF65, which is essential for splicing, U2AF35 is dispensable for in vitro splicing of some model pre-mRNAs containing strong Py tracts (i.e., a stretch of pyrimidines beginning at position-5 relative to the 3' splice site and extending 10 or more nucleotides upstream into the intron (Burge et al., 1999). The presence of U2AF35 and its interaction with U2AF65 was however found essential for in vitro splicing of a pre-mRNA substrate with a Py tract that deviates from the consensus (Guth et al., 1999). Introns with nonconsensual or weak Py tracts were previously called "AG-dependent" (Reed, 1989). Biochemical complementation experiments performed with extracts depleted of endogenous U2AF demonstrated that splicing of AG-dependent introns was rescued only when both U2AF subunits were added and not with U2AF65 alone (Zuo and Maniatis, 1996; Guth et al., 1999; Wu et al., 1999).
The importance of the small subunit of U2AF in vivo was first shown by the finding that the fruit fly Drosophila melanogaster ortholog of human U2AF35 (dU2AF38) is essential for viability (Rudner et al., 1996). Orthologues of U2AF35 are also essential for the viability of the fission yeast Schizosaccharomyces pombe (Wentz-Hunter and Potashkin, 1996) and the nematode Caenorhabditis elegans (Zorio and Blumenthal, 1999b) and for the early development of zebrafish (Golling et al., 2002). Two more recent studies in Drosophila further provided hints of a role for dU2AF38 in splicing regulation. First, loss-of-function mutations in dU2AF38 affected splicing of the pre-mRNA encoding the female-specific RNA-binding protein Sex-lethal (Nagengast et al., 2003). Second, depletion of the small subunit of U2AF by RNA interference (RNAi) affected alternative splicing of the Dscam gene transcript (Park et al., 2004).
To determine whether U2AF35 plays any vital role in mammalian cells, we used RNAi to specifically ablate its expression in HeLa cells. Because in higher vertebrates primary transcripts encoding U2AF35 can themselves be alternatively spliced to generate two protein isoforms termed U2AF35a and U2AF35b (Pacheco et al., 2004), we designed small inhibitory RNA (siRNA) duplexes specifically to knockdown expression of each isoform. Our results reveal an essential function of the major U2AF35a isoform in cell division and suggest that both U2AF35 protein isoforms act as regulators of alternative splicing of a specific subset of genes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The sequences used to target each U2AF35 isoform were as follows: h35a: 5'-CCA UUG CCC UCU UGA ACA UdTdT-3' and h35a': 5'-CCC UCA AAA CUC UUC CCA GdTdC-3', which target the U2AF35a isoform-specific exon 3 (GenBank accession number NM_006758, nt172–190 and nt200–218, respectively), and h35b: 5'-CCA UCU UGA UUC AAA ACA UdTdT-3' and h35b': 5'-UCC CCA AAA CAG UGC ACA GdAdC-3', which target the U2AF35b isoform-specific exon Ab (GenBank accession number AJ627978, nt164–182 and nt192–210, respectively). siRNA targeting Ski-interacting protein (SKIP) sequence was 5'-AGG GUA UGG ACA GUG GAU UdTdT-3' (GenBank accession number NM_012245, nt1274–1292). Synthetic sense and antisense oligonucleotides (BioSpring, Frankfurt, Germany) were annealed in 100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4), 2 mM magnesium acetate for 1 min at 95°C and 1 h at 37°C and frozen. In a typical siRNA transient transfection, 35-mm-diameter Petri dishes were seeded with 6 x 104 cells on the day before transfection. Twenty-four hours later, siRNA duplexes were transfected using 5 µl Lipofectin (Invitrogen) according to the supplier's recommendations (150 nM duplex siRNA per well).
Microscopic Analysis
To analyze cell viability, cells were trypsinized, washed in phosphate-buffered saline (PBS), and resuspended in 0.2% trypan blue. Apoptosis was assayed using the In situ Cell Death detection kit (Roche Diagnostics, Indianapolis, IN). To determine the mitotic index, cells were trypsinized, fixed, and then made to adhere to coverslips. For immunofluorescence microscopy, cells grown on coverslips were rinsed briefly in PBS, fixed in 3.7% formaldehyde:PBS for 10 min at room temperature, and washed with PBS. Cells were then permeabilized with 0.5% (wt/vol) Triton X-100:PBS for 10 min at room temperature, and washed with PBS. DNA was either stained with the vital dye Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) or with TO-PRO-3 iodide (Molecular Probes, Eugene, OR). Immunofluorescence and confocal microscopy were performed as previously described (Custodio et al., 2004). The following primary antibodies were used: mouse monoclonal antibodies directed against 
-tubulin (clone B-5-1-2; Sigma), cyclin B and Cdc27 (clone 18 and clone 35, respectively; BD Biosciences PharMingen, San Diego, CA), and a human serum directed against the kinetochore proteins CENP-A/C (kindly provided by Professor W. van Venrooij, Nijmegen, The Netherlands). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Flow Cytometry
Cell cycle analysis was performed by flow cytometry in accordance with standard protocols. Cells were harvested and fixed in ice-cold 70% (vol/vol) ethanol for 2 h at 4°C. Before flow cytometry, fixed cells were washed in PBS, resuspended in DNA staining solution (propidium iodide [PI, 20 µg/ml]; RNAse A [1 mg/ml]; 0.1% [vol/vol] Triton X-100 in PBS), and incubated at room temperature for 30 min. DNA content was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using the CellQuest software (BD Biosciences). Cell aggregates were gated out of the analysis based on the width of the PI fluorescence signal and each profile was obtained from 20,000 gated events. The percentage of G1, S, and G2/M phase populations were calculated with ModFitLT cell cycle analysis software package (Verity Software House, Topsham, ME).
Immunoblotting
Total cell protein extracts were prepared by scraping cells into SDS-PAGE buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% 
-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) with 200 U/ml benzonase (Sigma- Aldrich), incubating for 10 min at room temperature, and then boiling for 5 min. For Western blot analysis, 15 µg of total protein extract was separated on 8, 10, or 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Western blotting was carried out following standard procedures. The following primary antibodies were used: mouse monoclonal antibodies directed against -tubulin (clone B-5-1-2; Sigma), Cdc27 and Cdc25B (clone 35 and clone 23, respectively; BD Biosciences PharMingen), and rabbit polyclonal serums directed against -actin (provided by Prof. Ira Herman, Tufts University, Boston, MA) and U2AF35 (kindly provided by Prof. Tom Maniatis, Harvard University, Cambridge, MA). Immunoblots were developed using horseradish peroxidase–coupled secondary antibodies and detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Microarray Analysis
At 48 h after siRNA treatment, 107 cells were harvested, and total RNA was extracted. Hybridization and scanning of human U133A GeneChip Arrays (Affymetrix, Santa Clara, CA) were performed as recommended by the manufacturer. The complete list of genes induced or repressed by each siRNA treatment is presented in Supplementary Table 1.
RT-PCR and Real-Time Quantitative PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen), and treated with RNase-free DNase I (Roche Diagnostics). RT-PCR reactions were random primed and cDNA was produced using Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. PCR products were separated by gel electrophoresis and detected by ethidium bromide staining. All primer sequences are presented in Supplementary Table 2. qRT-PCR reactions were performed in the ABI7000 Sequence Detector (Applied Biosystems, Foster City, CA). The relative abundance of each gene was calculated from standard curves of CT versus quantity of mRNA obtained from a serial dilution of cDNA produced from human universal RNA (Applied Biosystems, Bulletin 2) and using U6 small nuclear RNA as an endogenous reference. The relative expression of different isoforms of the same gene was calculated using a derivative of the 2–
Ct method as described (Schmittgen et al., 2003). qRT-PCR analysis of U2AF35a and U2AF35b expression was performed as previously described (Pacheco et al., 2004) using GADPH as an endogenous reference (TaqMan gene expression assay 4333764T, Applied Biosystems, Foster City, CA) and the noninterfered sample as the calibrator.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). At least two different protein isoforms are derived from the human U2AF1 gene: U2AF35a, the translation product of a primary transcript containing exon 3, and U2AF35b, which arises from an alternatively spliced transcript in which exon Ab replaces exon 3 with no alteration of the reading frame (Figure 1A). The U2AF35a isoform is 9–18-fold more abundant than U2AF35b, depending on the tissue analyzed (Pacheco et al., 2004). In this study, we designed siRNA duplexes specifically to knockdown expression of either the U2AF35a isoform containing exon 3 (h35a) or the U2AF35b isoform containing exon Ab (h35b; Figure 1A). Analysis by BLAST search confirmed that the selected sequences were unique to U2AF35. Two different oligonucleotides were used to target each isoform (see Materials and Methods), and similar results were obtained with both. As a negative control, we used the GL2 dsRNA, which targets the firefly luciferase gene (Elbashir et al., 2001a).
|
). This confirmed the efficient and specific reduction of each mRNA isoform relative to the levels in mock-transfected cells (Figure 1C). Western blot analysis of control mock-transfected cells showed a single band corresponding to U2AF35 (Figure 1D, lane 1). After treatment with siRNAs, however, two closely migrating bands were detected (Figure 1D, lanes 2–4): the major, lower band corresponded to the most abundant protein isoform a, whereas the minor, upper band corresponded to the less abundant protein isoform b. Because isoform a is expressed at much higher level, treatment with siRNAs directed against U2AF35a caused a significant decrease in the lower band intensity (Figure 1D, lanes 2 and 4), whereas siRNAs directed against U2AF35b almost abolished expression of the minor, upper band (Figure 1D, lanes 3 and 4).
From these data we conclude that siRNA can specifically repress the expression of U2AF35a and U2AF35b isoforms at both the mRNA and protein levels.
Depletion of U2AF35a Inhibits Cell Proliferation and Causes Cell Death by Apoptosis
To determine whether U2AF35 proteins are essential, we transfected HeLa cells with GL2 (negative control), h35a and h35b siRNAs and analyzed them 24, 48, and 72 h after transfection. The number of viable and nonviable cells was counted after staining with trypan blue. The results show that knockdown of either U2AF35a or U2AF35b impaired cell proliferation (Figure 2A); however, knockdown of U2AF35a, alone or in conjunction with U2AF35b, had a more drastic effect on cell number than knockdown of U2AF35b alone. We further observed that at 72 h after transfection, knockdown of U2AF35a reduced the proportion of viable cells to 64%, whereas knockdown of U2AF35b had no significant effect when compared with treatment with the control siRNA (Figure 2B). We further observed that the fraction of apoptotic cells increased about threefold in U2AF35a-depleted cells, whereas depletion of U2AF35b had no significant effect on apoptosis (Figure 2C). Interestingly, simultaneous knockdown of U2AF35a and U2AF35b induced more cell death than knockdown of U2AF35a alone (Figure 2C), arguing that U2AF35b may play an additional accessory role important for cell survival.
|
To further investigate the effects of depleting U2AF35 on the cell cycle, RNAi-treated cells were stained with a DNA dye. Cells with decondensed chromosomes were scored as interphase, whereas cells at any stage from prophase to telophase were scored as mitotic. As shown in Figure 2E, the proportion of mitotic cells increased from 6.0 ± 0.6% of control cells to 12.4 ± 0.9% of cells transfected with h35a and to 16.7 ± 2.74% of cells transfected with h35a and h35b siRNAs. Knockdown of U2AF35b alone did not significantly affect the mitotic index (Figure 2E). To further study the effect of U2AF35 on mitosis, we analyzed living cells growing on grided coverslips 24, 48, and 72 h after transfection with the same siRNAs. Rounded cells with condensed chromosomes and a smooth, uniform membrane were scored as mitotic, and their positions were noted on a coverslip replica grid. After 45 min, the same cells were reobserved. The total number of cells that had progressed through cytokinesis and those that were still in mitosis was estimated. As depicted in Figure 2F, knockdown of U2AF35a, but not of U2AF35b, induces an increase in the proportion of cells that do not progress through cytokinesis, suggesting a mitotic delay.
To determine more accurately at which phase of mitosis the cells depleted of U2AF35a were delayed, double-labeling experiments were performed using a DNA dye to visualize the chromosomes and antibodies against -tubulin to visualize spindle microtubules or against kinetochore protein CENP-A/C to visualize the site of attachment of the spindle microtubules to the chromosomes (Figure 3A). The proportion of cells in different mitotic stages was estimated (Figure 3B). In both control and U2AF35b-depleted cells, most mitotic cells were in metaphase when all chromosomes and kinetochores are aligned in the center of a bipolar spindle. By contrast, most mitotic cells in populations depleted of U2AF35a (either alone or in conjunction with U2AF35b) have chromosomes and kinetochores distributed all along an abnormally elongated spindle. To identify this mitotic stage unambiguously, we performed immunofluorescence microscopy using antibodies to B-type mitotic cyclins (Figure 3A), which are degraded at the metaphase–anaphase transition (Murray, 2004). The mitotic cells depleted of U2AF35a and with unaligned chromosomes still contained B-type cyclins, indicating that they were most probably still in prometaphase, when chromosomes shuffle back and forth between the poles and the center of the spindle. In parallel, knockdown of U2AF35a alone or in conjunction with U2AF35b induced a significant decrease in the proportion of mitotic cells in metaphase, anaphase, and telophase (Figure 3B), as expected if cells were delayed (or arrested) in prometaphase. Our data additionally show that simultaneous knockdown of both U2AF35a and U2AF35b induced the appearance of mitotic cells with a monopolar spindle (Figure 3A, d and h). These cells were only detected at late time points after siRNA treatment (60–72 h), when they represent up to 13% of the mitotic cells (Figure 3C).
|
Expression Profile Analysis of Cells Depleted of U2AF35a and U2AF35b Isoforms
A possible explanation for the cell division phenotypes observed is that depletion of U2AF35 leads to impaired pre-mRNA processing of genes essential for cell division, in turn resulting in depletion of the corresponding mRNAs and proteins. To further explore this possibility, we performed a genome-wide analysis of the effect of depleting U2AF35a and U2AF35b on the expression of the HeLa cell transcriptome. Affymetrix oligonucleotide microarrays representing 14,500 well-characterized human genes were used. The relative abundance of each transcript was analyzed 48 h after transfection with siRNAs targeting U2AF35a, U2AF35b, or both. As reference samples, total RNAs were isolated from mock-transfected cells. Two independent RNAi experiments were performed and the efficiency of U2AF35 depletion was confirmed by RT-PCR. When U2AF35a was knocked down, the levels of 572 mRNAs were significantly different from the reference sample (i.e., at least 1.5-fold different in the two independent experiments). Most mRNAs (n = 525; 92%) were underrepresented, whereas 47 (8%) were overrepresented (Supplementary Table 1, A and B, respectively). In contrast, when U2AF35b was knocked down, only 138 mRNAs were changed (73% underrepresented and 27% overrepresented; see Supplementary Table 1, C and D, respectively). Simultaneous depletion of U2AF35a and U2AF35b affected the levels of 378 mRNAs (92% underrepresented and 8% overrepresented; see Supplementary Table 1, E and F, respectively). To highlight the differences in mRNA expression patterns in cells depleted of each U2AF35 isoform, we selected mRNAs that were at least twofold different in the cells depleted of one isoform when compared with the reference cells and analyzed their expression levels in the cells that were either depleted of the other isoform or simultaneously depleted of both isoforms (Figure 4, A and B). Thus, Figure 4, A and B, depicts subgroups of the mRNAs listed in Supplementary Table 1, for which more stringent cutoff values were used. The vast majority of mRNAs (>90%) altered in cells depleted of U2AF35a were also affected in cells depleted of both U2AF35a and U2AF35b, but their levels remained largely unchanged in cells depleted of U2AF35b (74%, <1.5-fold difference). From all mRNAs altered in cells depleted of U2AF35b, 25 (46%) were also affected in cells depleted of U2AF35a (Figure 4B). Most of the mRNAs whose expression was altered specifically by either U2AF35a or U2AF35b knockdown and whose genes could be identified corresponded to functional groups such as cell cycle and cell proliferation genes, signal transduction and transcription factors, DNA damage and stress response genes, or metabolic pathway components. However, neither the over- nor the underrepresented transcripts displayed any striking functional clustering (see Supplementary Table 1).
|
In summary, microarray analysis revealed that knockdown of each U2AF35 isoform affects expression of distinct mRNAs.
U2AF35a Is Required for Efficient Splicing of a Subset of Endogenous Transcripts
Previous evidence indicated that U2AF35 is required for splicing of pre-mRNA substrates that contain AG-dependent introns (Guth et al., 1999, 2001; Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999a). We therefore reasoned that mRNAs underrepresented in the microarray analysis of U2AF35-depleted cells might correspond to these pre-mRNA substrates that fail to be efficiently spliced and consequently are retained and degraded in the nucleus. To test this hypothesis, we searched among the transcripts that were specifically and consistently underrepresented in U2AF35- depleted cells for pre-mRNAs that contain introns with nonconsensus 3' splice sites and that also encode proteins functionally relevant for cell proliferation and cell cycle control. Using these criteria, we selected 11 introns from five different genes (Figure 5A). For all these genes, the level of spliced mRNA was found underrepresented in cells depleted of U2AF35a (Figure 5B). Splicing of each intron was analyzed by semiquantitative RT-PCR of total RNA with specific primers targeting the flanking exons (primer sequences are described in Supplementary Table 2). On the basis of early studies, we searched for an increase in ratio of unspliced to spliced RNA (Pikielny and Rosbash, 1985). The results are shown in Figure 6 and summarized in Figure 5A. From all introns tested, we only detected changes in intron 11–12 (i.e., the intron between exons 11 and 12) from Cdc27 and intron 12–13 from PNKP. Cdc27 is an essential subunit of the anaphase-promoting complex (APC), which is an ubiquitin-protein ligase responsible for the metaphase to anaphase transition and thus for exit from mitosis (Harper et al., 2002), and the PNKP gene encodes a polynucleotide kinase/phosphatase implicated in DNA repair after ionizing radiation or oxidative damage (Jilani et al., 1999).
|
|
). Western blotting and immunofluorescence microscopy further revealed a specific reduction of Cdc27 protein levels in U2AF35a-depleted cells (Figure 6, C and D). Taken together, these results suggest that knockdown of U2AF35a impairs splicing of a subset of pre-mRNAs, leading to decreased levels of the corresponding mRNAs and proteins.
Depletion of Either U2AF35a or U2AF35b Alters Alternative Splicing of Cdc25 Transcripts
Recently, an RNAi study in cultured Drosophila cells revealed that reducing the levels of both U2AF subunits modulated alternative splicing (Park et al., 2004). This prompted us to investigate a potential role of U2AF35 as regulator of alternative splicing in HeLa cells. To monitor alternative splicing, we chose to analyze the Cdc25B and Cdc25C genes, which play a key role in mitosis (Donzelli and Draetta, 2003) and encode well-described alternatively spliced isoforms (Baldin et al., 1997; Forrest et al., 1999; Wegener et al., 2000). Moreover, both genes contain nonconsensus 3' splice sites, and Cdc25B was found underrepresented in the microarray analysis of U2AF35a-depleted cells.
To study the effect of U2AF35 on alternative splicing of Cdc25B and Cdc25C transcripts, we performed semiquantitative RT-PCR and quantitative real time PCR. Primers for RT-PCR were designed based on exons 5 and 7 of Cdc25B to detect isoforms B2, B3, and B4 as products of 126, 249, and 354 bp, respectively (Figure 7A). To analyze Cdc25C expression, primers were designed based on exons 2 and 7 to yield three different products of about 522, 400, and 303 bp (Figure 7B). The major 522-bp band corresponds to the Cdc25C1 variant, and the 303-bp product represents the Cdc25C5 isoform. The 400-bp product corresponds to both Cdc25C3 and Cdc25C4 variants, because these cannot be distinguished based on their size; we therefore refer to this band as C3/C4.
|
To determine whether the observed effects on alternative splicing are specific for U2AF35a, we performed RNAi knockdown of the SKIP protein. SKIP was chosen because it was recently identified as a splicing factor required for cell division in HeLa cells (Kittler et al., 2004). The efficiency of the SKIP knockdown experiments was confirmed by Western blotting. As shown in Figure 7, A and B, depletion of SKIP did not affect the ratios of the alternatively spliced isoforms of both Cdc25B and Cdc25C relative to control cells. To further exclude the possibility that the observed changes in alternative splicing of Cdc25B and Cdc25C transcripts were a nonspecific response to impaired cell cycle progression, we analyzed RNA samples isolated from HeLa cells treated with nocodazole, a microtubule-destabilizing drug that arrests mitosis at prometaphase (Jordan et al., 1992). RNA was extracted from cells incubated in the presence of 60 ng/ml nocodazole for 6 and 16 h and analyzed by semiquantitative RT-PCR (Figure 7, E and F). In clear contrast to the effect of knockdown of U2AF35, nocodazole treatment had no significant effect on the ratio of alternatively spliced variants of Cdc25B (Figure 7E) and Cdc25C (Figure 7F) transcripts.
In conclusion, these data argue that both U2AF35 isoforms can function as regulators of alternative splicing in human cells.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), S. pombe (Wentz-Hunter and Potashkin, 1996), C. elegans (Zorio and Blumenthal, 1999b), and zebrafish (Golling et al., 2002). However, before this study it was unclear whether U2AF35 plays any vital role in mammalian cells.
Previously, we have shown that primary transcripts encoding splicing factor U2AF35 in higher vertebrates are alternatively spliced (Pacheco et al., 2004). Here, we used RNAi to specifically ablate the expression of U2AF35a and b isoforms in human HeLa cells. RNAi is currently used extensively to inhibit expression of specific genes in the cells of several organisms, including mammals (Huppi et al., 2005). By exploiting this evolutionarily conserved process, a specific gene can be silenced by a siRNA complementary to the mRNA product of the gene, which anneals to the mRNA and triggers its degradation. The assumption in any RNAi experiment is that the siRNA will selectively target its complementary mRNA for degradation; however, recent reports suggest that siRNAs can also nonspecifically alter the expression of a significant number of genes involved in diverse cellular functions (Jackson et al., 2003; Semizarov et al., 2003; Persengiev et al., 2004). To minimize nonspecific effects, we used two different oligonucleotides to target each U2AF35 isoform, and similar phenotypic effects were observed with both.
Our results show that knockdown of U2AF35a abrogated cell proliferation (Figure 2A), decreased cell viability to 64% (Figure 2B), increased about threefold the fraction of apoptotic cells (Figure 2C), and caused a massive delay in mitotic progression (Figure 2F). Importantly, more than 80% of depleted mitotic cells failed to progress through cytokinesis during a 45-min observation period, whereas the steady sate proportion of G2/M phase cells increased only from 14 to 22% in U2AF35a-depleted cells. This is probably because the mitotically arrested cells dye by apoptosis and become therefore excluded from the population. On the basis of these results, we conclude that U2AF35a plays an essential role in cell division.
Strikingly, an siRNA screen for genes required for HeLa cell division identified several components of the splicing machinery, namely SNRPA1, SNRPB, SNW1/SKIP, DHX8, DDX5, LSM6, and SART1 (Kittler et al., 2004). The phenotypes observed after knockdown of these splicing proteins included mitotic arrest and spindle abnormalities (Kittler et al., 2004). Moreover, conditional mutations in several splicing factors in yeast induce cell cycle arrest at the restrictive temperature (Dahan and Kupiec, 2004 and references therein) and genetic inactivation of splicing factor ASF/SF2 induced cell cycle arrest and apoptosis in chicken B-cells (Li et al., 2005). Thus, cell cycle arrest appears to be a common phenotype induced by depletion of splicing proteins. An involvement of RNA and RNA-binding proteins in mitotic spindle assembly was recently discovered (Blower et al., 2005), raising the possibility that some splicing factors may have an as yet unidentified direct role in cell division. Alternatively (or additionally), depletion of splicing proteins may indirectly lead to abnormal pre-mRNA processing of transcripts encoding proteins essential for cell division. Consistent with the latter hypothesis, in U2AF35a-depleted cells we detected an almost twofold increase in the ratio between unspliced and spliced transcripts encoding Cdc27, a twofold decrease of Cdc27 mRNA and correspondingly reduced Cdc27 protein levels (Figures 5B and 6, A, C, and D). Cdc27 is an essential subunit of the APC responsible for the metaphase-to-anaphase transition (Harper et al., 2002), and therefore reduced levels of Cdc27 protein are expected to prevent metaphase cells from progressing to anaphase. However, we found that U2AF35a-depleted cells accumulated predominantly in prometaphase, arguing that U2AF35a is probably required for efficient splicing of several pre-mRNAs, and the observed phenotype results from the cumulative effect of many reduced proteins.
Because we did not use splicing-specific microarrays, we could not identify genome-wide effects on splicing caused by loss U2AF35a. However, we detected reduced Cdc27 and PNKP mRNA levels both by microarray analysis and qRT-PCR, and we showed an increased ratio between the corresponding unspliced and spliced transcripts induced by U2AF35a depletion. These observations support the view that several underrepresented mRNAs in the microarray analysis may result from impaired splicing.
In a recent study, RNAi-mediated depletion of the essential splicing factor SF3a was shown to produce 90% cell death and cause a general block in splicing (Tanackovic and Kramer, 2005). On the basis of our results showing that depletion of the major U2AF35a isoform caused 40% cell death and affected splicing of only a subset of the transcripts analyzed, we conclude that human U2AF35 is not a constitutive splicing factor in vivo.
Although alternative splicing is well known to be regulated by RNA-binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA, a recent study using RNAi in Drosophila cells has unexpectedly shown that reducing the levels of core components of the spliceosome can affect alternative splicing (Park et al., 2004). These core splicing factors included snRNP proteins, DExH/D-box proteins and both subunits of U2AF (Park et al., 2004). Consistent with that finding, we show here that depletion of U2AF35a in HeLa cells specifically altered the ratios of alternatively spliced isoforms of transcripts encoding the cell cycle phosphatases Cdc25B and Cdc25C. Importantly, the Cdc25B phosphatase is essential for mitotic entry (Lindqvist et al., 2005) and is thought to play an oncogenic role (Galaktionov et al., 1995). In particular, the Cdc25B2 splice variant (which increased after depletion of U2AF35a) is not detected in nonneoplastic lymphoid tissues but is progressively up-regulated in indolent and aggressive human non-Hodgkin's lymphomas (Hernandez et al., 2000) and has also been shown to be differentially regulated in colorectal carcinomas (Hernandez et al., 2001). We therefore speculate that changing the levels of U2AF35 in a cell may promote the production of Cdc25B splice variants with different oncogenic potential.
The finding that knockdown of U2AF35b causes minor effects when compared with knockdown of U2AF35a is not surprising given that U2AF35b is 9–18-fold less abundant than U2AF35a in mammalian tissues (Pacheco et al., 2004). However, U2AF35b has been highly conserved throughout evolution, suggesting that this protein plays an important role in metazoans (Pacheco et al., 2004). Accordingly, we observe that U2AF35b depletion interferes with cell proliferation and augments the defects caused by knockdown of U2AF35a. Furthermore, microarray analysis revealed that loss of U2AF35b specifically altered the expression level of 138 mRNAs, and qRT-PCR experiments demonstrated that U2AF35b can specifically influence alternative splicing of Cdc25C transcripts.
In conclusion, this study establishes a link between the most abundantly expressed U2AF35a isoform and cell division and provides evidence suggesting that both the major and minor U2AF35 isoforms act as regulators of alternative splicing in human cells.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-01-0036) on July 19, 2006.
Address correspondence to: Maria Carmo-Fonseca (carmo.fonseca{at}fm.ul.pt)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Blower, M. D., Nachury, M., Heald, R., Weis, K. (2005). A Rae-1 containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell 121, 223–234.[CrossRef][Medline]
Burge, C. B., Tuschl, T. H., Sharp, P. A. (1999). Splicing precursors to mRNAs by the spliceosomes. In: The RNA World, ed. R. F. Gesteland, T. R. Cech, J. F. Atkins. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 525–560.
Custodio, N., Carvalho, C., Condado, I., Antoniou, M., Blencowe, B. J., Carmo-Fonseca, M. (2004). In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei. RNA 10, 622–633.
Dahan, O. and Kupiec, M. (2004). The Saccharomyces cerevisiae gene CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 gene. Nucleic Acids Res 32, 2529–2540.
Donzelli, M. and Draetta, G. F. (2003). Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Rep 4, 671–677.[CrossRef][Medline]
Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.[CrossRef][Medline]
Elbashir, S. M., Lendeckel, W., Tuschl, T. (2001b). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15, 188–200.
Forrest, A. R., McCormack, A. K., DeSouza, C. P., Sinnamon, J. M., Tonks, I. D., Hayward, N. K., Ellem, K. A., Gabrielli, B. G. (1999). Multiple splicing variants of cdc25B regulate G2/M progression. Biochem. Biophys. Res. Commun 260, 510–515.[CrossRef][Medline]
Galaktionov, K., Lee, A. K., Eckstein, J., Draetta, G., Meckler, J., Loda, M., Beach, D. (1995). CDC25 phosphatases as potential human oncogenes. Science 269, 1575–1577.
Golling, G., et al. (2002). Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat. Genet 31, 135–140.[CrossRef][Medline]
Guth, S., Martinez, C., Gaur, R. K., Valcarcel, J. (1999). Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65). Mol. Cell. Biol 19, 8263–8271.
Guth, S., Tange, T. O., Kellenberger, E., Valcarcel, J. (2001). Dual function for U2AF(35) in AG-dependent pre-mRNA splicing. Mol. Cell. Biol 21, 7673–7681.
Harper, J. W., Burton, J. L., Solomon, M. J. (2002). The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev 16, 2179–2206.
Hernandez, S., et al. (2001). Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma. Lab. Invest 81, 465–473.[Medline]
Hernandez, S., et al. (2000). cdc25a and the splicing variant cdc25b2, but not cdc25B1, -B3 or -C, are over-expressed in aggressive human non-Hodgkin's lymphomas. Int. J. Cancer 89, 148–152.[CrossRef][Medline]
Huppi, K., Martin, S. E., Caplen, N. J. (2005). Defining and assaying RNAi in mammalian cells. Mol. Cell 17, 1–10.[CrossRef][Medline]
Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G., Linsley, P. S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol 21, 635–637.[CrossRef][Medline]
Jilani, A., Ramotar, D., Slack, C., Ong, C., Yang, X. M., Scherer, S. W., Lasko, D. D. (1999). Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage. J. Biol. Chem 274, 24176–24186.
Jordan, M. A., Thrower, D., Wilson, L. (1992). Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J. Cell Sci 102, Pt 3401–416.[Medline]
Jurica, M. S. and Moore, M. J. (2003). Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14.[CrossRef][Medline]
Kittler, R., et al. (2004). An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036–1040.[CrossRef][Medline]
Li, X., Wang, J., Manley, J. L. (2005). Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation. Genes Dev 19, 2705–2714.
Lindqvist, A., Kallstrom, H., Lundgren, A., Barsoum, E., Rosenthal, C. K. (2005). Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome. J. Cell Biol 171, 35–45.
Merendino, L., Guth, S., Bilbao, D., Martinez, C., Valcarcel, J. (1999). Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG. Nature 402, 838–841.[CrossRef][Medline]
Mitchell, P. and Tollervey, D. (2001). mRNA turnover. Curr. Opin. Cell Biol 13, 320–325.[CrossRef][Medline]
Murray, A. W. (2004). Recycling the cell cycle: cyclins revisited. Cell 116, 221–234.[CrossRef][Medline]
Nagengast, A. A., Stitzinger, S. M., Tseng, C. H., Mount, S. M., Salz, H. K. (2003). Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. Development 130, 463–471.
Pacheco, T. R., Gomes, A. Q., Barbosa-Morais, N. L., Benes, V., Ansorge, W., Wollerton, M., Smith, C. W., Valcarcel, J., Carmo-Fonseca, M. (2004). Diversity of vertebrate splicing factor U2AF 35, identification of alternatively spliced U2AF1 mRNAs. J. Biol. Chem 279, 27039–27049.
Park, J. W., Parisky, K., Celotto, A. M., Reenan, R. A., Graveley, B. R. (2004). Identification of alternative splicing regulators by RNA interference in Drosophila. Proc. Natl. Acad. Sci. USA 101, 15974–15979.
Persengiev, S. P., Zhu, X., Green, M. R. (2004). Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18.
Pikielny, C. W. and Rosbash, M. (1985). mRNA splicing efficiency in yeast and the contribution of nonconserved sequences. Cell 41, 119–126.[CrossRef][Medline]
Reed, R. (1989). The organization of 3' splice-site sequences in mammalian introns. Genes Dev 3, 2113–2123.
Rudner, D. Z., Kanaar, R., Breger, K. S., Rio, D. C. (1996). Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects. Proc. Natl. Acad. Sci. USA 93, 10333–10337.
Schmittgen, T. D., Teske, S., Vessela, R. L., True, L. D., Zakrajsek, B. A. (2003). Expression of prostate specific membrane antigen and three alternatively spliced variants of PSMA in prostate cancer patients. Int. J. Cancer 107, 323–329.[CrossRef][Medline]
Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N., Fesik, S. W. (2003). Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. USA 100, 6347–6352.
Tanackovic, G. and Kramer, A. (2005). Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo. Mol. Biol. Cell 16, 1366–1377.
Wegener, S., Hampe, W., Herrmann, D., Schaller, H. C. (2000). Alternative splicing in the regulatory region of the human phosphatases CDC25A and CDC25C. Eur. J. Cell Biol 79, 810–815.[CrossRef][Medline]
Wentz-Hunter, K. and Potashkin, J. (1996). The small subunit of the splicing factor U2AF is conserved in fission yeast. Nucleic Acids Res 24, 1849–1854.
Wu, S., Romfo, C. M., Nilsen, T. W., Green, M. R. (1999). Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature 402, 832–835.[CrossRef][Medline]
Zamore, P. D., Patton, J. G., Green, M. R. (1992). Cloning and domain structure of the mammalian splicing factor U2AF. Nature 355, 609–614.[CrossRef][Medline]
Zorio, D. A. and Blumenthal, T. (1999a). Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans. Nature 402, 835–838.[CrossRef][Medline]
Zorio, D. A. and Blumenthal, T. (1999b). U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans. RNA 5, 487–494.[Abstract]
Zuo, P. and Maniatis, T. (1996). The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev 10, 1356–1368.
This article has been cited by other articles:
|
|
 |
|
  R. S. C. Cosme, Y. Yamamura, and Q. Tang Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus J. Virol., April 1, 2009; 83(7): 2839 - 2850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Andersen and N. Tapon Drosophila MFAP1 Is Required for Pre-mRNA Processing and G2/M Progression J. Biol. Chem., November 7, 2008; 283(45): 31256 - 31267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Grosso, A. Q. Gomes, N. L. Barbosa-Morais, S. Caldeira, N. P. Thorne, G. Grech, M. von Lindern, and M. Carmo-Fonseca Tissue-specific splicing factor gene expression signatures Nucleic Acids Res., September 1, 2008; 36(15): 4823 - 4832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Izquierdo Fas Splicing Regulation during Early Apoptosis Is Linked to Caspase-mediated Cleavage of U2AF65 Mol. Biol. Cell, August 1, 2008; 19(8): 3299 - 3307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rino, J. M. P. Desterro, T. R. Pacheco, T. W. J. Gadella Jr., and M. Carmo-Fonseca Splicing Factors SF1 and U2AF Associate in Extraspliceosomal Complexes Mol. Cell. Biol., May 1, 2008; 28(9): 3045 - 3057. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
400 cells per experiment). (D) Cell cycle analysis by FACS. (E) Proportion of mitotic cells (n 
