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J. Biol. Chem., Vol. 280, Issue 13, 12397-12404, April 1, 2005
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¶**
From the
Department of Genetics and Molecular Biology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary, Institute of Biochemistry, Biological Research Center, Temesvari körut 62, H-6726 Szeged, Hungary, and || Institute of Gene Biology, Russian Academy of Sciences, Vavilov Street 34/5, Moscow 117 334, Russia
Received for publication, August 12, 2004 , and in revised form, January 31, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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220 amino acid long C-terminal regions of the proteins, and as compared with the 315 amino acid yeast protein, the TGS1 homologs of higher eukaryotes possess an additional extended N-terminal region. Here, we describe the Drosophila melanogaster TGS1, which we identified on the basis of its affinity to bind RNA. D. melanogaster TGS1 is translated via an internal translation start site from an RNA, which has coding capacity for two polypeptides. We present genetic evidence that tgs1 is an essential gene in Drosophila and that its mutation compromises RNA m3G cap formation. Identification of the tgs1 mutation in Drosophila opens up the possibility of a detailed analysis of the complex function(s) in which TGS1/PIMT participates in higher eukaryotes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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FixII genomic library through consecutive hybridization steps with 32P-labeled cDNA probes.
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The plasmids pET3a-DTL, pET3a-DTLu, and pET3a-DTLd were constructed in order to express DTL proteins in bacteria. For this reason, the dtl cDNA and two subfragments containing either the uORF or the dORF (encompassing the regions from nucleotides +80 to +739 and from +613 to +2008) were inserted into pET3a (Novagen). Recombinant proteins were produced in BL21(DE3) cells following isopropyl 1-thio-
-D-galactopyranoside induction according to standard protocols.
For the construction of DTL-enhanced green fluorescent protein (EGFP) fusions, dtl genomic DNA fragments were inserted in front of the gene encoding the EGFP in plasmids pEGFP-N3 and pEGFP-N1 (Promega). Plasmids pDTLu-EGFP and pDTLus-EGFP both contain the region from +80 (the second ATG of dtl mRNA, see Figs. 1 and 4) to +739 inserted in front of EGFP (the numbers indicate the position of the A of ATGs unless otherwise indicated). The reading frames (RFs) of dORF and of EGFP in pDTLu-EGFP are in phase. pDTLus-EGFP was constructed from pDTLu-EGFP by filling up the BssHII site at nucleotide 502, which resulted in a +1 shift in the RF, such that in pDTLus-EGFP the RF from +80 to the end of EGFP is open. pDTLd-EGFP carries the region from +614 (from the seventh ATG of dtl mRNA) to +739 inserted between the cytomegalovirus promoter and EGFP-coding region, such that the RF of dORF and EGFP are in phase. In pDTLud-EGFP, a fragment encompassing the region from +80 to +502 is inserted in front of the EGFP, such that the phase of EGFP is the same as in uORF. In pDTL-EGFP, the dtl-coding region from nucleotide +80 to +1803 is inserted in-frame in front of the EGFP-coding region. The structures of the plasmid constructs were verified by sequencing. Details on the constructions are available upon request.
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-galactosidase activity were selected, and the Drosophila cDNA segment they carried was recovered and sequenced. The cDNAs we selected represented an as yet unidentified Drosophila gene that we designated dtl (Drosophila Tat-like).
Fly Stocks, Generation, and Rescue of dtl Mutants—Fly stocks were maintained at 25 °C on standard food. The line l(3)S096713 (967) was identified in the Szeged Drosophila Stock Center, and the position of the P-element insertion was determined by nucleotide sequencing following plasmid rescue. Deficiency 189 (d189) was isolated by identifying improper excision after remobilization of the P-element present in l(3)S096713. The P-element was mobilized by the p(
2–3) transposase source, and mutations were isolated by scoring for loss of the "mini-white" marker. Imprecise excision lines were identified by genetic crosses based on their phenotype over Df(3R)P14 and further analyzed by Southern blotting of genomic DNA from hemizygotes and by PCR using primers derived from the 31-bp terminal inverted repeat sequence of the P-element and flanking genomic sequences. Finally, the breakpoints of the deletion were determined by sequencing. To generate dtl transgene, a 4.6-kb genomic fragment corresponding to the region between –926 and +3700 (with respect to the transcription initiation site of dtl) was inserted into the CaSpeR4 vector (pCaSpeR-DTL) (6), which has the mini-white+ selectable marker. Modified versions of the above plasmid, pCaSpeR-DTLum and pCaSpeR-DTLdm (Fig. 1), were constructed by inserting a synthetic oligonucleotide (CTAGTCTAGACTAG) with stop codons (underlined) in all three reading frames at position +502 and by deleting the region between +871 and +1868, respectively. For the construction of dtl transgene with a single amino acid change in the MTase active center (pCaSpeR-DTLS423R), a G to C point mutation resulting in a Ser to Arg amino acid change at amino acid position 423 was generated in pCaSpeR-DTL by PCR using the mutagenic primers 5'-TTTCCTGAGGCCTCCGTGG-3' and 5'-CCACGGAGGCCTCAAGGAAA-3' in combination with external primers. The mutation generated a new StuI restriction site (AGGCCT), which was used to verify the construct and the cDNA synthesized on it, following RT-PCR. Transformants were made by P-element-mediated germ-line transformation of w1118 host embryos using helper plasmid p2–3 as described by Spradling (7). Independent mini white+ transformant lines carrying single insertions were isolated, and the insertions were localized to chromosomes by using balancers. From each construct, at least two independent transformed lines were established and used in this work. To analyze the dtl phenotype, y w; d189/TM3,Sb Ser y+ females were crossed to yw; P967/TM3, Sb Ser y+ males. To determine the lethal phase, the numbers of animals reaching the second or third instar or pupal stage were recorded. d189/P967 animals and their siblings carrying the TM3 balancer were separated based on the y and y+ markers. The development of dtl mutant and control siblings was followed, and the time needed for 50% of the animals to molt to the next instar was taken as characteristic. At least five independent experiments, each involving 30–60 animals, were performed.
Immunological Techniques—The DTL protein and its derivatives were expressed in bacteria. For antibody production, the fragment of the dtl cDNA corresponding to the region from +1243 to + 1612 (encoding amino acids 236–357 of DTLd) was generated by NcoI digestion and inserted in-frame into pET3a to obtain pET3a-DTLnn. The truncated DTL protein expressed in pET3a-DTLnn-containing Escherichia coli BL21(DE3) cells was isolated from inclusion bodies, refolded, and injected into rabbits. Antibodies were purified on a protein A-Sepharose affinity column. Protein electrophoresis was performed according to standard protocol. After the transfer of proteins by electroblotting, the membranes were blocked for 1 h in 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and incubated overnight with primary antibody diluted in TBST. Membranes were washed, incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (DACO), washed again extensively, and developed using the ECL (Amersham Biosciences) kit by following the manufacturer's recommendations.
Anti-m3G monoclonal antibody (Ab-1, Oncogene) was used to detect the m3G cap in Drosophila tissues by immunostaining and for immunoprecipitation. For immunostaining, larvae in various stages of development were dissected in phosphate-buffered saline and fixed in 10% formaldehyde, 1 mM EGTA solution. Treatment with anti-m3G primary antibody at 4 °C was followed by anti-mouse fluorescein isothiocyanate-conjugated secondary antibodies (DACO) for 2 h at room temperature.
For immunoprecipitation, 10 µg of total RNA samples isolated from wild-type and dtl mutant L3 larvae, as indicated in Fig. 6, were incubated with protein A-Sepharose-bound m3G-specific monoclonal antibody, collected, washed, and treated with proteinase K. Supernatants were treated similarly without washes. Proteinase K-treated supernatants and precipitates were extracted with phenol-chloroform and precipitated with ethanol. For Northern hybridization, immunoprecipitated and control RNA samples were separated on 8% denaturing polyacrylamide gel, blotted to nylon membrane by capillary transfer, and hybridized with 32P-labeled probes.
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-32P]UTP. Tissue Culture and Transfections—To assess DTL expression in eukaryotic cells, plasmids carrying DTL-EGFP fusions were transfected into HeLa cells. 3–5 µg of purified DNA was transfected from each plasmid into 106 cells by the calcium phosphate coprecipitation method, as described previously (8). 40 h after transfection, cells were harvested, observed under a microscope, and divided into two fractions. One fraction was sorted by fluorescence-activated cell sorter to estimate the transfection efficiency and green fluorescent protein expression. The other fraction was lysed, the protein concentration was determined by the Bradford method, and the proteins were analyzed on denaturing polyacrylamide gel. The proteins were blotted onto nitrocellulose membrane, and Western blots were developed with a green fluorescent protein-specific antibody (a generous gift of Dr. Ferenc Nagy). Blots were developed using the ECL kit (Amersham Biosciences) as recommended and exposed to x-ray films.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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4–6 kDa larger than EGFP and EGFP, whereas pDTLd-EGFP directed only EGFP production (Fig. 4B, lanes 1 and 2). This result suggested that, as in bacteria, translation initiation in eukaryotic cells also started at the ATG located at the start of dORF (at +538) and that no fusion product of uORF-dORF-EGFP was produced. To test whether translation occurring on the uORF had any effect on the translation of dORF, we constructed additional EGFP fusions. In pDTLud-EGFP, the EGFP-coding region was fused in phase to the region coding for the N-terminal part of uORF at position 502. This plasmid has the ATG opening the uORF of dtl but lacks the ATG at the start of the dORF. In pDTLus-EGFP, the reading frame within the uORF was altered by the insertion of four nucleotides at position +502. Consequently, in this last construct, the ATG at +80 opens a reading frame extending to the end of the EGFP-coding region. HeLa cells transfected with pDTLud-EGFP produced no fusion protein but EGFP. In contrast, in extracts of pDTLus-EGFP-transfected cells, we detected proteins identical to those seen in pDTLu-EGFP, e.g. EGFP and a 4–6-kDa larger fusion protein. These results led us to conclude that translation of the dtl message starts at an internal translation initiation site located at 538 nucleotides from the 5' end of the message. Accordingly, the major product of dtl is a 491 amino acid protein. Western blots of Drosophila cell extracts developed by antibodies raised against bacterially expressed DTL support this result by showing the production of a 60-kDa protein (Fig. 5A). However, it must be noted here that our antibodies also detected other immunoreactive bands (both larger and smaller) in animals in different developmental stages (data not shown).
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Dtl Has an Essential Function in Drosophila—For the functional analysis of dtl, we initiated genetic studies to identify and characterize dtl mutation. A lethal P-element insertional mutant D. melanogaster line, (3)S096713 P967, identified in an independent screen, carries a P-element insertion 35 bp downstream from the transcription initiation site of dtl (Fig. 1B). P967 homozygotes and P967 mutation over deficiency Df(3R)P14, which uncovers the 90FG region, are lethal. The P-element insertion in P967 is only 107 nucleotides upstream from the transcriptional start of the adjacent ada2a/rpb4 gene, and consequently, the function of ada2a/rpb4 could also be affected by the insertion (9). Because P-element insertions are often hypomorphic, in order to facilitate further genetic studies, we generated an additional dtl allele by remobilizing the P-element in line l(3)S096713. In line d189 isolated by this technique, improper excision of the P-element resulted in a deletion extending from +35 to –1235 with respect to the transcription start site of dtl (Fig. 1). In P967/d189 second instar larvae, we could not detect wild-type dtl-specific mRNAs by RT-PCR analysis. On Western blots of protein extracts of P967/d189 L1 larvae, a reduced amount of the 60-kDa protein recognized by anti-DTLd polyclonal antibody was detected (data not shown). We believe that the DTLd protein present in P967/d189 animals at L1 represents a fraction of the maternally provided DTLd still present in this stage of development.
The development of P967/d189 animals is nearly normal until they reach stage L3. The earliest difference we observed between dtl mutants (P967/d189) and their control siblings (TM3 heterozygotes) was that the mutants needed a slightly longer time to complete the L2 instar (23.1 ± 1.2 h versus 26.5 ± 0.9). Nonetheless, 84.6% P967/d189 animals completed the larval stages of development and formed puparium. However, none of them emerged as an adult, all perishing in stages P3-P4. To provide definite proof that the observed phenotype of P967/d189 animals resulted from the lack of dtl function, we constructed and analyzed Drosophila lines carrying dtl transgenes. A 4.6-kb genomic fragment corresponding to the dtl-coding region and adjacent chromosomal DNA was inserted into pCaSpeR4 to generate pCaSpeR4-DTL (Fig. 1), which was injected into embryos. Following appropriate genetic crosses, two independent transgene carrier lines (P967/d189 P[DTL]) were established. The two lines gave identical results in the following experiments. The P[DTL] transgene completely restored the wild-type phenotype of P967/d189 animals. Thus, we established that the early pupal lethality observed in P697/d189 animals is a result of dtl mutation.
Loss of dtl Results in a Loss of m3G Cap-containing RNA Pool—The strongest homology that DTLd protein displays with proteins characterized so far relates to TGS1 of yeast and mammals in its C-terminal region (4). To ascertain whether the loss of enzyme activity needed for m3G cap formation of small non-coding RNAs could be detected in dtl mutants, we compared the immunostaining of dissected larval tissues of wild-type and dtl mutant (P967/d189) animals with m3G cap-specific antibody. The immunostaining of tissues of larvae in different stages of development with m3G-specific monoclonal antibody indicated that, in dtl animals, the pool of m3G cap-containing RNAs gradually diminished. As assessed by the staining of dissected larval tissue with m3G-specific antibody, by the end of the third larval stage, shortly before the mutant animals perished, virtually no m3G cap-containing RNAs were detected (Fig. 6A). By comparing the RNAs of wild-type and mutant animals immunoprecipitated with m3G-specific antibody, we also observed the loss of m3G cap-containing snRNAs from dtl (d189/967) mutants. Northern blot hybridization to immunoprecipitated samples of total RNA isolated from third instar larvae revealed a decrease in the level of m3G cap-containing U2 and U4 snRNA (Fig. 6B). The immunostaining of 189/967 P[DTL] larvae demonstrated that a transgene carrying the entire dtl region P[DTL], which rescued the phenotype, also restored the m3G cap-containing RNA pool (data not shown). We next considered whether the loss of m3G cap and lethality were linked and resulted from the loss of dtl uORF or dORF or both. To answer these questions, we further tested transgenes for their ability to rescue the dtl phenotype (pupal lethality) and restore the m3G capping of snRNAs. Transgenes P[DTLum] and P[DTLdm] are modified versions of P[DTL], generated by the insertion of stop codons in the uORF (at nucleotide 502) and the deletion of a region of the dORF (between nucleotides 871 and 1868), respectively. Animals carrying transgene P[DTLdm]inthe d189/967 background display a phenotype identical to that seen in d189/967 (pupal lethality) and the loss of m3G cap-containing RNAs as determined by either immunostaining or immunoprecipitation (Fig. 6, A and B). In contrast, in animals carrying transgene P[DTLum] in an identical genetic background, we detected m3G cap-containing RNAs at a similar level as in wild-type animals by both immunostaining and immunoprecipitation (Fig. 6, A and B). However, although the P[DTLum] transgene rescued P3-P4 lethality, this rescue was not complete and animals carrying the transgene died as pharat adults or immediately after hatching. Interestingly, the transgenes P[DTLdm] and P[DTLum] together in trans position resulted in a complete rescue, leading to the development of fertile adult animals without obvious defects. These data suggest that the product of dtl downstream ORF (DTLd) is TGS1, the fission yeast homolog of which has been demonstrated recently to catalyze methyl transfer from AdoMet to M7GTP (10), and that the function determined by this ORF is essential for Drosophila development. Nevertheless, the loss of a function other than that of TGS1 should also be considered because the product of DTL dORF is structurally more similar to its mammalian than to its yeast homolog and the mammalian counterpart has been shown to have a transcriptional coactivator function (1). Accordingly, we set out to test whether the lethality is a result of the loss of TGS1 activity or any other function of DTLd. For this reason, we generated a dtl transgene with a single amino acid change at the MTase active site, P[DTLS423R]. An identical mutation of the yeast homolog has been reported to abolish enzyme activity (4). The mutation also generated an StuI cleavage site, which we used to verify that the mutant TGS1 mRNA was expressed in the established transgenic lines (data not shown). Insertion of the P[DTLS423R] transgene did not change the lethal phase of d189/967 animals. Similarly, no m3G cap-containing RNAs can be detected in d189/967 P[DTLS423R] animals by immunostaining (Fig. 6A). From these data, we concluded that the TGS1 encoded by the downstream ORF of the dtl gene of Drosophila is essential for normal fly development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The second ORF of dtl has a coding capacity for a protein of 491 amino acids. Each cDNA fragment that we isolated in the screen was from this part of the gene and contained the region corresponding to amino acids 200–350. As we isolated the dtl cDNAs based on the RNA binding ability of the encoded peptides, we assume that this region carries the RNA binding motif of DTLd. This part of the protein includes an Arg-rich amino acid sequence KKKRRQRQI similar to the RNA-binding region of HIV TAT (RKKRRQRRR) (11, 12).
The relationship between the structural organization of the two ORFs of dtl resembles the features of complex transcription units of retroviruses and retrotransposons where a specific translational switch often ensures the synthesis of gag-pol polyproteins from one polycistronic RNA. In retroviruses, among them HIV, the utilization of two partially overlapping ORFs requires a –1 ribosome slip (13, 14). We addressed the question of whether there is any evidence of a similar mechanism at the overlapping start/stop codons of the dtl message. Because signals directing ribosome slip at overlapping codons within viral RNAs are effectively recognized by the bacterial translational machinery (15), we first studied the translation of the dtl message in bacteria. As shown in Fig. 3, Western blot analysis of dtl proteins expressed in E. coli, indicated that, whereas both ORFs of the dtl cDNA were translated in bacteria, no cotranslation of the two ORFs occurred and translation initiation of the second ORF started at an internal AUG. In summary, these experiments allowed the conclusion that, under the conditions tested, a ribosome slip resulting in fusion protein formation did not occur in E. coli. In eukaryotic cells, under the conditions tested, the first ORF was either not translated or was translated with such low efficiency that its product was not detectable. Plasmid constructs containing the entire uORF and the first part of dORF fused to the EGFP-coding region directed the synthesis of a protein only a few kDa larger than EGFP itself (Fig. 4). This result suggests that translation of the messages synthesized from the fusion genes started only at internal AUGs. Frameshift mutations within the uORF did not affect the synthesis of the fusion protein, indicating that translation of the upstream part of the message is not a prerequisite for initiation at an internal AUG.
Initiation of translation at a downstream AUG recently has been described in a number of eukaryotic messages and appears to be far more frequent than previously believed (16–18). Although our knowledge of the mechanisms is far from complete, it is well accepted that upstream AUGs, upstream ORFs, and internal ribosome entry sites (IRESs) often play critical roles in the expression of genes encoding key regulatory proteins (19, 20). In Drosophila, the activity of the IRESs of some genes is developmentally controlled (21). The internal initiation at IRESs is believed to be augmented by RNA-binding proteins. However, the roles of protein factors suspected of being involved are largely unknown. This together with other uncertainties concerning the mechanism prompted Kozak (22) to suggest the redefinition of IRESs as "internal regulatory expression sequences."
The mechanism by which translation of the dORF of dtl at an internal AUG of the message is initiated is unclear. A high degree of secondary structure in the 5' region of dtl is not predicted, suggesting that the presence of a stable secondary structure does not inhibit ribosome scanning. Surprisingly, in HeLa cells transfected with plasmids carrying DTL-EGFP fusions, we detected the production of a protein, which we believe is the authentic EGFP. One reason for EGFP expression in the reporter constructs could be that, in the presence of a dtl upstream region, ribosomes recognize AUGs within the message at high efficiency. Whether this is a result of a structural feature of the 5' region of the dtl message or a translated peptide from the 5' region remains to be clarified.
Homology searches for relatives of the 491 amino acid major product of dtl in the databases revealed that the closest homolog of the Drosophila protein is Tgs1, the 315 amino acid RNA-hypermethylase first identified in S. cerevisiae (3). Yeast Tgs1 and DTL exhibit 41% identity and 58% similarity in their C-terminal 200 amino acid regions. This is the evolutionarily conserved MTase catalytic domain present in Tgs1-like proteins of other organisms as well (4). The large N-terminal domain present in DTL is absent from yTgs1 but is present in Tgs1 orthologs from animals and plants. The related mammalian protein is hTgs1, also identified as PIMT, an 852 amino acid nuclear receptor coactivator-interacting protein (1). Besides the homology between PIMT/hTgs1 and DTL in their C-terminal regions, they also display limited similarity in their central regions. Although DTL clearly has RNA binding affinity and its RNA binding motif is similar to the Arg-rich RNA binding motifs present in many RNA-binding proteins (among others, HIV Tat), we believe that this similarity is coincidental and that no functional homology exists between DTL and HIV TAT.
By remobilization of a P-element (P967) integrated in the vicinity of dtl TSS, we generated a dtl allele d189, which lacks the entire dtl regulatory region. P967/d189 animals are early pupa lethal, demonstrating that dtl has an essential function. Mutant animals need a slightly longer time than their siblings for completion of the L2 stage. The severe phenotype, early pupal lethality, seen in the absence of dtl is in contrast with the mild phenotype observed in the absence of Tgs1 in S. cerevisiae (3). Immunohistochemical staining with an antibody specific for m3G cap indicated that, in dtl animals as well as in TGS1 mutant yeast cells, the pool of RNAs with m3G cap is reduced compared with the wild-type controls. The loss of TGS1 activity results in pupal lethality of dtl animals (d189/967). The fact that the loss of MTase activity is the underlying cause of lethality is clearly indicated by the failure of transgenes mutated in the MTase active site to rescue the phenotype. The difference in severity of the mutations of dtl and its yeast counterpart yTgs1 might also reflect functional differences between the m3G cap-containing RNAs in the two organisms. One intriguing question that arises is whether the loss of TGS1 in dtl mutants leads to lethality because of a general failure of vital functions such as splicing and rRNA maturation in which snRNA and small nucleolar RNAs are involved or because of depletion of a specific type of m3G cap-containing RNA. Our immunoprecipitation-hybridization studies demonstrated that RNA samples obtained from dtl animals shortly before they died contained practically no m3G-capped U2 and U4 snRNAs. In accord with this finding, immunostaining of larval tissues also indicated a nearly complete loss of the pool of m3G cap-containing RNAs. The last residual m3G cap-specific antibody-reactive material that we could observe was in imaginal histoblasts (Fig. 6). We believe that 967/d189 animals are dtl null and that the DTLd protein present in mutants in L1 represents a fraction of the maternally provided DTLd still present at this stage of development. However, we cannot exclude the possibility that the 967 allele is hypomorphic. Thus, the roles of maternal and zygotically expressed TGS1 in the survival of dtl animals cannot be determined from our data. Even so, it is surprising that, as can be judged from the immunostaining results, a considerable amount of the m3G cap-containing RNA pool is present in 967/d189 mutants even at a late larval stage. Our data do not permit a conclusion regarding whether the loss of TGS1 activity in a higher eukaryote such as Drosophila is lethal due to a general failure of RNA metabolism or because of its effect on one or a few specific type(s) of small RNA(s). Nonetheless, the essential role of the enzyme is underlined by the observation that a single amino acid change in the MTase domain results in lethality. As the product of dtl, dORF is more similar in its structure to the mammalian PIMT protein than it is to the yeast TGS1, which has only the MTase domain. It is also conceivable that the enzyme in higher eukaryotes has a function requiring MTase activity other than modification of the small RNA cap structure. To our surprise, dtl function can be provided by a combination of two transgenes carrying intact uORF and dORF, neither of which alone is capable of complete rescue. The most probable explanation for this observation could be that the two ORFs of dtl complement each other in trans. Whether the complementation results from the interaction of two proteins translated from the two ORFs of dtl or from the interaction of RNA and protein molecules remains to be clarified. At present, we cannot exclude the possibility that the upstream region of dtl itself has a function other than influencing the production of the major DTLd protein. Genetic and molecular studies are underway to disclose the relationship that exists between the two products of the dtl gene.
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FOOTNOTES |
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¶ Present address: Hematopoesis Department, University of Maryland, Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855.
** To whom correspondence should be addressed. Tel.: 36-62-544686; Fax: 36-62-544651; E-mail: borosi{at}bio.u-szeged.hu.
1 The abbreviations used are: PIMT, peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain; Mtase, methyltransferase; AdoMet, S-adenosyl-L-methionine; m3G, 2,2,7-trimethylguanosine; TGS1, trimethylguanosine synthase 1; DTL, Drosophila Tat-like; RF, reading frame; ORF, open reading frame; dORF, downstream ORF; uORF, upstream ORF; EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; TSS, transcription start site; HIV, human immunodeficiency virus; d189, deficiency 189; RACE, rapid amplification of cDNA ends; sn, small nuclear; TAR, transactivation response element.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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