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Rebecca L. Read,2
James Pearce,1
Chris J. Norbury,3 and
Shao-Win Wang1*
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, United Kingdom,1 Cancer Research UK Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom,2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom3
Received 25 October 2005/ Returned for modification 18 November 2005/ Accepted 14 December 2005
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ABSTRACT |
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INTRODUCTION |
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In addition to the canonical nuclear enzyme, eukaryotic cells contain several unconventional PAPs. In a study of the fission yeast Schizosaccharomyces pombe we previously identified the cid1 gene family, which encodes a distinct group of PAPs conserved from yeast to humans (46). In Caenorhabditis elegans, the Cid1-related protein GLD-2 was characterized as a cytoplasmic PAP (42), and Xenopus laevis GLD-2 was subsequently found to be required for CPEB-mediated cytoplasmic polyadenylation during early development (3). Kwak et al. (20) recently described five human orthologs of the Cid1 protein, named Hs1 to Hs5. Among these, Hs4 is responsible for the synthesis of long poly(A) tails in human mitochondria (37). Sequence comparisons identify six members of the S. pombe cid1 gene family (cid1, cid11, cid12, cid13, cid14, and cid16) (46). Cid1 (for caffeine-induced death suppressor), the first member of this family to be described, was identified through its ability when overexpressed to confer resistance specifically to the combination of hydroxyurea and caffeine, which overrides the replication checkpoint (44). Cid13 was identified independently based on its ability, when overexpressed, to rescue the hydroxyurea sensitivity of checkpoint rad mutants (29, 31). In addition to its related role in checkpoint control, Cid12 is required for faithful chromosome segregation and RNA interference-mediated heterochromatin assembly at centromeres (24, 46). Purified recombinant Cid1 catalyzed polyadenylation in vitro (29), as did Cid13-myc immunoprecipitated from S. pombe (31). Both Cid1 and Cid13 are cytoplasmic proteins, indicating that their function is likely to be distinct from that of the nuclear PAP. It has been proposed that Cid13 acts to extend the poly(A) tails of ribonucleotide reductase mRNA to counteract deadenylation and enhance protein synthesis (31).
In Saccharomyces cerevisiae the Cid1-related protein Trf4 (which has a redundant homolog, Trf5) was reported to have DNA polymerase activity in vitro (47). Subsequently, immunoprecipitates of Trf4-hemagglutinin (HA) from S. cerevisiae were found to have PAP, but not DNA polymerase, activity (31). However, unlike most of the Cid proteins, Trf4 and Trf5 are nuclear (12) and are proposed to function in a nuclear RNA surveillance system, whereby the Trf4-mediated oligoadenylation of RNAs targets them for rapid degradation by the exosome. This resembles the role of polyadenylation in bacterial RNA turnover (15, 21, 40, 51).
In this study, we describe the characterization of fission yeast Cid14, a functional homologue of S. cerevisiae Trf4/5. We present evidence that Cid14 is required for the polyadenylation of rRNAs as a prelude to their degradation by the exosome. Polyadenylation mediated by Cid1-related proteins therefore appears to have different consequences and to affect diverse functions, depending on the subcellular localization and identity of the target RNA.
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MATERIALS AND METHODS |
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Plasmids and site-directed mutagenesis. Plasmid pCB427 (TRF4 URA3 CEN) was kindly provided by M. F. Christman (7). Plasmids for overexpression of cid1 and cid14 under the control of the GAL1 promoter were constructed by PCR amplification of the cid1+ and cid14+ open reading frames from S. pombe genomic DNA using the primer pairs CGCGGATCCGCGAATATGAACATTTCTTCTGCAC and ATAGTTTAGCGGCCGCATTCTTATTCACTCAGAATTGTCACCATC and CGCGGATCCGCGGAGATGGGTAAAAAAAGCGTGTC and ATAGTTTAGCGGCCGCATTCTTATCTAAAAACGTTTGCGTATTTTTTTCGC, respectively, followed by digestion with BamHI and NotI and subsequent ligation into BamHI- and NotI-cleaved pYES2 (Invitrogen), generating pYES2cid1 and pYES2cid14. pREP1cid14 was constructed by PCR amplification of the cid14 open reading frame from S. pombe genomic DNA using the primer pair CID14F (TTTGGATCCCATGGGTAAAAAAAGCGTGTCATTTAACCGC) and CID14R (TTTGGATCCCTAAAAACGTTTGCGTATTTTTTTCGCTCG), followed by digestion with BamHI and subsequent ligation into BamHI-cleaved pREP1. PCR using primers CID14R and DADAF (CCAAATTATATTTACCTACTTCAGCTCTTGCTCTTGTAATAATATCACCTGAG) and primers CID14F and DADAR (CTCAGGTGATATTATTACAAGAGCAAGAGCTGAAGTAGGTAAATATAATTTGG) was used to generate the cid14 open reading frame in two fragments overlapping by 53 bp, with the region of overlap spanning codons 298 and 300, which were altered in the primer sequence to encode alanine rather than the aspartate residues specified by the wild-type gene at these positions. The resulting fragments were mixed and used in a secondary PCR with primers CID12F and CID12R. After digestion with BamHI, the final product was ligated into pREP1 to generate pREP1cid14DADA. All plasmid constructions were confirmed by complete sequencing of the inserts using an ABI sequencer and ABI PRISM dRhodamine reagents (Applera UK).
Gene disruption. The one-step disruption method was used following PCR-mediated generation of the entire ura4+ gene flanked by 80-bp segments from the 5' and 3' regions of cid14+ using oligonucleotides TGGTTCAATTAGTCATATATTGATTTTTATAAGAATTCTACAAGATAGGTATAGAAAACCATTCTATCCATGTCTTAGAGAAATCCCACTGGCTATATGT and ACATT TTAACATATGCTCACGATGTTGACGAACCCCTTCATCAACTGATAAT ATGGTACTTAGGATACTAGGGAAATCTTAATTCTAAATGCCTTCTG AC. Construction of the chromosomally HA- and green fluorescent protein (GFP)-tagged cid14 strains (cid14-HA and cid14-GFP) was accomplished by an analogous method using primers CGGAAGACGAAGTACCTATTATTGAGGACACCACTGCTTCAGATGAAGAATCTCGAGCGAAAAAAATACGCAAACGTTTTCGGATCCCCGGGTTAATTAA and AAAATTACTACAGATCCATAGTGAGTTTAAGGTGTTGATTAATTTATTACTGGTATTCGAGTTT ACTGGCTAAATCGTAAGAATTCGAGCTCGTTTAAAC. The cid14::LEU2 allele was generated by a secondary one-step disruption of the cid14::ura4+ allele using primers as described previously (46).
Antibodies and immunoblotting. Cell extracts were prepared by trichloroacetic acid precipitation following glass bead disruption (6). Immunoblotting was performed essentially as described elsewhere (2). The mouse anti-influenza virus HA monoclonal antibody HA-11 (Babco, Berkeley, CA) was used for detection of HA-tagged Cid14 proteins. Cdc2 was detected using the mouse monoclonal antibody Y100 (generated by J. Gannon and kindly provided by H. Yamano). Horseradish peroxidase-conjugated anti-mouse antibodies (Sigma, Poole, United Kingdom) and enhanced chemiluminescence (ECL; Amersham) were used to detect bound antibody.
Gel filtration chromatography.
Chromatographic separation of S. pombe lysate from a 100-ml culture of the cid14-HA strain in midexponential growth was carried out using a Superose-6 column attached to an fast protein liquid chromatography workstation (Amersham Pharmacia) as previously described (45). The eluate was collected in 0.5-ml fractions, and these were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The column was calibrated using the standards dextran blue (
2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), ß-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa).
Microscopy. Visualization of GFP in living cells, embedded in 0.6% low-melting-point agarose after staining with Hoechst 33342 (5 µg/ml), was performed at room temperature as previously described (45). Images were acquired using a Zeiss Axioplan 2 microscope equipped with a Planapochromat 100x objective, an Axiocam cooled charge-coupled device camera, and Axiovision software (Carl Zeiss, Welwyn Garden City, United Kingdom) and were assembled using Adobe PhotoShop.
RNA isolation and quantitative PCR.
Total RNA was isolated by hot-phenol extraction and purified using RNeasy (QIAGEN). Total S. pombe RNA (0.5 µg) was reverse transcribed (SuperScript; Invitrogen) using an oligo(dT) primer according to the manufacturer's instructions. Quantitative PCR was performed using SYBR green PCR Master Mix (Applera UK) and primer pairs specific for ste11 (ATACCTGGAGAAGCGACGAA and GAATGAGCGGCGAACTCATA), mei2 (TATGGACGAGAACCCTGCTT and GACGTGCATTATTGGGAGCA), and cdc2 (normalizer gene; CCAGCTAGTGAACGGTGTAA and TCCGCAAAGGGACACCAAAT). Samples were run in triplicate on the Applied Biosystems 7300 real-time PCR system (Applera UK). Relative quantities of transcript were determined using the 2–
Ct formula, where Ct is defined as the cycle at which fluorescence is determined to be statistically above background, Ct is the difference in Ct of the gene of interest and Ct of the normalizer gene, and Ct is the difference in Ct of the strain tested and Ct of the control cells.
PCR poly(A) test. Polyadenylation tests were performed according to the rapid amplification of cDNA ends-polyadenylation technique (RACE-PAT) method as described previously (32) using a reverse transcription-PCR (RT-PCR) kit (SuperScript; Invitrogen) with oligo(dT) anchor primer AT (GCGAGCTCCGCGGCCGCGT12) and a primer specific for 25S rRNA (25F1: GCGGCTGCCTGACAAGGCAATGCCGCGGAG), 5.8S rRNA (5.8F1: TCAGCAACGGATCTCTTGGCTCTCGCATCG), or 5S rRNA (5F1: GTTCCCGTCCGATCACTGCAGTTAAGCGTC). PCR products were cloned (TOPO TA cloning; Invitrogen) and sequenced using an ABI sequencer and ABI PRISM dRhodamine reagents (Applera UK). For detection of mature rRNA, primer pairs 25F2 (GAAGATGGGCGATGGTTGATGAAACGGAAGTG) and 25R1 (ACAAATCTTGGGAACAAAGGCTTAATCTCAGCAG), 5.8F1 and 5.8R1 (AATGACACTCAAACAGGCATGCCTTTGGTAG), and 5F1 and 5R1 (AGCCTACAGCACCCCGGATTCCCATGTTGT) were used for 25S, 5.8S, and 5S rRNA, respectively.
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RESULTS |
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double mutant with cid14+ under the control of a galactose-inducible promoter. As shown in Fig. 1A, cid14+ complemented the temperature-sensitive growth phenotype of the trf4ts top1 double mutant. The double mutant containing vector alone grew at 25°C but was unable to grow at 36°C. Expression of TRF4 or cid14+ but not cid1+ was able to support growth of the double mutant at both temperatures. We conclude that the S. pombe cid14+ and S. cerevisiae TRF4 genes are functionally homologous. Multiple sequence comparisons suggest that TRF4 and TRF5 were generated by a relatively recent gene duplication event, so it is likely that the single cid14 gene performs the TRF4/5-like function in S. pombe. Consistent with this hypothesis, no synthetic lethality was observed in cid14 mutants in combination with deletion of any other cid genes (data not shown).
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cells grew with a doubling time of approximately 9 h, compared with 2.5 h for a cid14+ strain. In addition, unlike trf4 and trf5 mutants, cid14 deletion mutants remained fully viable on mutation of top1, which encodes the fission yeast topoisomerase I (41). Microscopic examination showed that cid14 cells retained an essentially normal morphology as judged by cell staining to reveal distribution of DNA, septa, and actin (Fig. 1C and data not shown).
Deletion of cid14 leads to an increased chromosome segregation failure rate but not precocious sister chromatid separation during metaphase arrest.
In S. cerevisiae Trf4 was reported to be required for the establishment of sister chromatid cohesion during S phase to ensure faithful chromosome segregation at mitosis (47). We therefore asked whether or not Cid14 is required for faithful chromosome segregation. To address this point, wild-type and cid14 strains containing an integrated GFP-tagged swi6 gene were constructed. GFP-Swi6 is localized in the nucleoplasm, giving a background of faint nuclear signal and several brightly fluorescent spots corresponding to the heterochromatic centromeres and telomeres (28). As shown in Fig. 2A and B, anaphase chromosome segregation in living cells as monitored by GFP-Swi6 fluorescence appeared normal in the cid14+ background. In contrast, abnormal anaphase progression (segregation of two unequal masses of GFP-Swi6) was observed in 35% of binucleate cid14 cells. Consistent with these apparent chromosome segregation defects, cid14 mutants had a moderate increase in the rate of minichromosome loss. Mean loss rates were 0.14% and 0.04% per division for cid14 and wild-type strains, respectively (average of three independent clones), using strains containing the nonessential ade6-M216-marked Ch16 minichromosome derivative of chromosome 3 (25). Despite the chromosome segregation defects described, cid14 mutants are viable, indicating that the spindle checkpoint might be required for the survival of these cells. Indeed, as shown in Fig. 2C, deletion of bub1 (4) but not mad2 (10) is lethal in cid14 cells, indicating that a subset of the spindle checkpoint factors are required for the survival of cid14 cells.
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cells after shifting to the restrictive temperature to induce arrest at metaphase (Fig. 2E). In contrast, cells displaying separated Ndc80-GFP signals (more than three spots) were dramatically increased in a cid12 mutant defective in centromeric cohesion mediated by the RNA interference machinery (24). Thus, unlike S. cerevisiae trf4 mutants, S. pombe cid14 cells showed no indication of premature sister centromere separation during metaphase arrest. Characterization of Cid14 protein in S. pombe. Targeted recombination was used to add an HA epitope tag sequence to the 3' end of the cid14 open reading frame in its normal chromosomal context, generating the cid14-HA strain. Western blot analysis showed that the Cid14 protein had an aberrant mobility when analyzed by SDS-PAGE. While the calculated molecular mass of the protein is 78 kDa, the Cid14 protein isolated from S. pombe cells migrated as a predominant band of approximately 100 kDa (Fig. 3A). Next, we examined the expression of the Cid14 protein during the cell cycle. For this, cdc25-22 cid14-HA cells, released synchronously after cell cycle arrest at the restrictive temperature, were collected every 25 min and their lysates were subjected to immunoblotting. Cell cycle synchrony and position were monitored by determining the septation index. As shown in Fig. 3B, the Cid14 protein level remains constant with no obvious mobility shift through the cell cycle. Recently, S. cerevisiae Trf4 was reported to function in a complex with Mtr4 and Air1/2 (21, 40, 51). To determine whether or not Cid14 also functions as part of a higher-order complex, we used gel filtration chromatography to examine the native size of S. pombe Cid14 in a cell lysate prepared from the cid14-HA strain. As shown in Fig. 3C, the cellular Cid14 protein was found to be almost exclusively included in a large complex with an apparent size between 669 and 2,000 kDa. This result indicates that Cid14, like Trf4, forms part of a large complex in vivo.
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strain (Fig. 1B). Examination of living cid14-GFP cells by fluorescence microscopy showed that Cid14-GFP (and, by inference, Cid14) was predominantly localized to the nucleus and was concentrated in a condensed spot in the nonchromosomal domain (Fig. 3D), previously assigned as the nucleolus (39). This phenomenon was further explored by examination of Cid14-GFP localization in an nda3-KM311 mutant in which chromosomes condense after incubation at the restrictive temperature of 20°C for 8 h. As shown in Fig. 3E, the intense fluorescence of Cid14-GFP signal was associated with the smallest chromosome, consistent with the fact that the shortest S. pombe chromosome (III) contains the rRNA gene clusters. In line with these results, Cid14 colocalized with the region of intense ethidium bromide staining (Fig. 3F), which distinguishes the nucleolus from the rest of the nucleus (38). The nucleolar concentration of Cid14-GFP was observed in cells from all stages of the cell cycle (Fig. 3D).
Aberrant nucleolar structures in cid14 mutants.
Next, we asked whether or not Cid14 deficiency might affect nucleolar architecture. Nucleolar structure was visualized in living cells using a protein that was a fusion between GFP and the nucleolar protein Gar2 (34). In wild-type cells, Gar2-GFP occupied roughly one-half of the nucleus, in a discrete region distinct from the bulk chromosomal DNA, against a background of fainter nuclear signal (Fig. 4A, lower panels). In contrast, a more diffuse Gar2-GFP fluorescence signal was observed in the majority of cid14 cells (examples 1 to 3) and, upon entry into mitosis, these cells often failed to segregate Gar2-GFP properly (example 4). This phenomenon was further explored by time-lapse microscopy. As shown in Fig. 4B, in contrast to wild-type cells, in which Gar2-GFP separated equally into the daughter cells at mitosis, abnormal anaphase progression (segregation of two unequal masses of Gar2-GFP) frequently occurred in cid14 cells. Taken together, these results suggest that Cid14 might play a role in the organization of the nucleolus to ensure faithful segregation during mitosis.
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and cid14 dis3-54 strains, suggesting that Cid14 plays a role in adenylating the rRNAs. In line with its predicted polyadenylation activity, the polyadenylated 25S rRNA products were found to be dependent on the conserved aspartate residues 298 and 230 in the nucleotide transferase motif (GS X10 DXD) of Cid14 that are known to be essential for the catalytic activity of this protein family (Fig. 5D). When expressed in the cid14 strain from the nmt1 promoter in the plasmid pREP1cid14DADA in the presence of thiamine, this mutant form of Cid14, unlike the wild-type protein, was unable to restore the polyadenylated 25S rRNA species in these cells.
Next, we asked whether or not the other three types of rRNA might be polyadenylated and accumulate in a dis3-54 mutant. As shown in Fig. 6A, similar results were observed in the RACE-PAT assay of 5.8S RNA. Sequencing analysis confirmed Cid14-dependent polyadenylation of 5.8S RNAs, as these products were dramatically reduced in the cid14 and cid14 dis3-54 strains. The residue products presented in cid14 cells were due to nonspecific priming as confirmed by sequencing of 17 randomly selected clones (data not shown). No obvious difference was observed in the amount of these polyadenylated 5.8S rRNA products in dis3-54 mutants compared with the wild-type strain. Although it has been reported that 18S RNAs are polyadenylated to a certain level (19), we were not able to detect any polyadenylated 18S rRNA products using an approach similar to that described above (data not shown).
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dis3-54 strain.
In line with these results, cid14 cells were hypersensitive to 5-fluorouracil (5-FU; Fig. 6G), a chemical which interferes with the degradation of rRNA precursors (9).
Deletion of cid14 leads to stimulation of meiosis in pat1-114 cells.
In C. elegans, the Cid1-related protein GLD-2 together with its RNA binding partner GLD-3 controls various aspects of germ line development, including the mitosis/meiosis decision (42), while in Xenopus xGLD-2 is required for CPEB-mediated polyadenylation-induced translation during oocyte maturation (3). We were therefore interested to see whether or not Cid14 plays a role in meiosis in fission yeast. To test this, we examined the genetic interaction between cid14 and pat1, which encodes a general inhibitor of sexual differentiation (22). As shown in Fig. 7A and B, whereas the pat1-114 single mutants enter meiosis at 30°C, the pat1-114 cid14 mutants initiated meiosis at the normally permissive temperature of 25°C. Similarly, pat1-114 dis3-54 mutants underwent meiosis at the lower temperature. These results suggest that Cid14 together with the exosome might play a role in meiosis, closely interacting with the Pat1 kinase.
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cells as well as in dis3-54 mutants grown at the permissive temperature 30°C. As shown in Fig. 7C, deletion of cid14, or dis3 mutation, led to accumulation of mei2 transcripts as determined by quantitative PCR. Mutation of dis3 also affected the stell mRNA level. The elevated levels of mei2 mRNA could account for the stimulation of meiosis in pat1-114 cid14 and pat1-114 dis3-54 cells. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mutants (data not shown). It has been reported that polyadenylation of several rRNA species in a rrp6 background depends on the nuclear poly(A) polymerase (19, 51). Whether or not Pla1, the canonical S. pombe poly(A) polymerase, is responsible for the polyadenylation of these transcripts remains to be determined. However, the significance of polyadenylation of these transcripts is unclear; it does not appear to stimulate their degradation by the exosome, as the mutation of dis3 has no influence on the amount of these transcripts. In line with these results, cells that lack a functional cid14+ gene grow slowly and are hypersensitive to 5-FU (Fig. 6G). Consistent with its role in rRNA metabolism, S. pombe Cid14 is a nucleolar protein and is required for maintenance of nucleolar structure (Fig. 3 and 4). Similarly, Dis3 protein (and, by inference the exosome) is enriched in the nucleolus (17). Intriguingly, polyadenylation of 5S rRNAs, transcribed by RNA polymerase III outside the nucleolus, is also dependent on Cid14 to a degree (Fig. 6). This could be due to the function of nucleolus in the processing of rRNAs and their subsequent assembly into preribosomes. The fact that a Trf4-dependent polyadenylation-assisted degradation mechanism is also responsible for the degradation of RNA polymerase II transcripts (51) broadens the implication of these results. Possible involvement of the nucleolus in mRNA export has been proposed, based on observations made in yeasts and higher eukaryotic cells (5, 8, 13, 33). These results suggest that the nucleolus might function as a site of general RNA quality control, although it is still unclear how the cell determines which transcripts should be degraded.
Chromosome segregation defects were another obvious feature of cid14-deficient cells (Fig. 2). In S. cerevisiae Trf4 was reported to be required for the establishment of sister chromatid cohesion during S phase to ensure faithful chromosome segregation at mitosis (47). However, unlike S. cerevisiae Trf4 mutants, S. pombe cid14 cells showed no indication of premature sister centromere separation during metaphase arrest (Fig. 2D and E). Given the function of Cid14 in rRNA metabolism, ribosome biogenesis and translation defects might have a direct effect on some aspect of chromosome structure or kinetochore function. Alternatively, the impact of a loss of Cid14 function in mitosis may be through impaired expression of genes required for faithful chromosome segregation. Intriguingly, in S. pombe, the dis3+ gene was first identified through a screen for mutants defective in mitotic chromosome separation (17, 26, 27). The same screen identified dis2+, which encodes a serine-threonine protein phosphatase identified as a component of the pre-mRNA cleavage and polyadenylation complex (30). Similar defects have been reported for pfs2 mutants, which are defective in another component of the pre-mRNA cleavage and polyadenylation complex (43). The characterization of S. pombe mutants defective in the 5'-3' exoribonuclease Dhp1 suggested an additional connection between RNA processing and chromosome segregation (35). It will be interesting to determine whether or not chromosome segregation defects are a general feature of mutants defective in RNA processing.
Our results also revealed a possible function of Cid14 in the control of meiosis. In C. elegans, the Cid1-related protein GLD-2 promotes entry into meiosis from the mitotic cell cycle (16). In contrast, deletion of cid14 leads to stimulation of meiosis in pat1-114 cells (Fig. 7). This effect could be due to the deprivation of nutrients as a consequence of defective ribosome biogenesis. However, pat1-144 cells arrested at G1 rather than entering meiosis after deprivation of nutrients by nitrogen starvation at the permissive temperature 25°C (data not shown). These results suggest that Cid14 together with the exosome might have a more direct role in meiotic control. Additional connections between RNA processing and meiosis were suggested by the identification of S. pombe pac1+, which encodes an RNase III-like RNase, as a multicopy suppressor of the uncontrolled meiosis driven by pat1 deficiency (14). The pac1 gene product probably inhibits mating and meiosis by degrading mRNAs required for sexual development. The accumulation of mRNAs required for meiosis in exosome mutants suggests that these transcripts might be constantly degraded by the exosome in mitotic cells to prevent ectopic meiosis. Experiments are under way to explore this possibility.
Cid1-like proteins control a variety of functions in S. pombe, C. elegans, and vertebrates. The identification of Cid14 as the functional homologue of S. cerevisiae Trf4/5 indicates the generality of a polyadenylation-assisted degradation mechanism, which is likely to be conserved in higher eukaryotes. Interestingly, in contrast to the situation in other organisms, no cytoplasmic function of Trf4/5 has been described. Studying the way in which Cid1-like proteins function in lower eukaryotes is expected to provide greater understanding of the biological functions of polyadenylation in eukaryotic cells in general.
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ACKNOWLEDGMENTS |
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This work was supported by Cancer Research UK and The Wellcome Trust (Research Career Development Fellowship to Shao-Win Wang).
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FOOTNOTES |
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Present address: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
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REFERENCES |
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