Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M506963200 on October 17, 2005

J. Biol. Chem., Vol. 281, Issue 2, 733-743, January 13, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/2/733    most recent
M506963200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, G.
Right arrow Articles by Davis, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, G.
Right arrow Articles by Davis, R. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Flatworm Spliced Leader 3'-Terminal AUG as a Translation Initiator Methionine*Formula

Guofeng Cheng{ddagger}, Leah Cohen§, David Ndegwa§, and Richard E. Davis{ddagger}§1

From the {ddagger}Departments of Pediatrics and Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045 and the §Department of Biology, City University of New York Graduate Center, College of Staten Island, Staten Island, New York, 10314

Received for publication, June 27, 2005 , and in revised form, September 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spliced leader (SL) RNA trans-splicing contributes the 5' termini to mRNAs in a variety of eukaryotes. In contrast with some transsplicing metazoan groups (e.g. nematodes), flatworm spliced leaders are variable in both sequence and length in different flatworm taxa. However, an absolutely conserved and unique feature of all flatworm spliced leaders is the presence of a 3'-terminal AUG. We previously suggested that the Schistosoma mansoni spliced leader AUG might contribute a required translation initiator methionine to recipient mRNAs. Here we identified and examined trans-spliced cDNAs from a large set of newly available schistosome cDNAs. 28% of the trans-spliced cDNAs have the SL AUG in-frame with the major open reading frame of the mRNA. We identified over 40 cDNAs (40% of the SL AUG in-frame clones) that require the SL AUG as an initiator methionine to synthesize phylogenetically conserved N-terminal residues characteristic of orthologous proteins. RNA transfection experiments using several schistosome stages demonstrated that the flatworm SL AUG can serve as a translation initiator methionine in vivo. We also present in vivo translation studies of the schistosome initiator methionine context and the effect of the spliced leader AUG added upstream and out-of-frame with the main open reading of recipient mRNAs. Overall, our data have provided evidence that another function of flatworm spliced leader trans-splicing is to provide some recipient mRNAs with an initiator methionine for translation initiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spliced leader (SL)2 RNA trans-splicing is an RNA processing event that forms the mature 5' end of mRNAs (1–3). The two substrates for the reaction are 1) a small RNA, the spliced leader RNA, and 2) a pre-mRNA. The spliced leader RNA consists of a small exon, the spliced leader, ranging in size from 15 to 52 nucleotides. The SL exon is followed by a 5' splice site and an intron without a 3' splice site. The recipient pre-mRNA substrate begins with an outron (an intron lacking a 5' splice site) followed by an exon or several exons and introns. Trans-splicing adds the spliced leader sequence to the 3' splice site of the outron, generating the mature 5' end of the mRNA. Spliced leader addition provides not only the 5'-terminal sequence but also brings a new cap to the mature mRNA. In metazoa, a trimethylguanosine (TMG) cap (m2,2,7GpppN) is present on the spliced leader RNA, and thus the mRNAs acquire a TMG cap (4–9).

Trans-splicing is present in some sarcomastigophora protozoa, hydra, nematodes, rotifers, flatworms, and early chordates (8–20). SL addition thus represents a major form of gene expression present in divergent groups of eukaryotes. For some organisms and genes, a function for trans-splicing is known. Trans-splicing in concert with 3' end formation serves to resolve polycistronic transcripts into monocistronic mRNAs for almost all genes in the kinetoplastida, ~20% of Caenorhabditis elegans genes, several genes in the early chordate Oikopleura, and at least one schistosome gene locus (11, 21–27). In C. elegans, two spliced leaders have been identified, SL1 and SL2 (17, 28). SL2 transsplicing participates in the resolution of polycistronic transcripts (21, 22, 24, 29). However, SL1 addition is responsible for the majority of trans-splicing in nematodes, accounting for 50–90% of trans-splicing in different nematodes. Notably, SL1 trans-splicing is not typically involved in resolution of polycistronic transcripts, and overall, its primary function remains unclear. Recently, we have shown that the addition of the TMG-capped SL1 to test transcripts does not have a significant effect on nematode mRNA stability and translation when compared with non-trans-spliced m7G-capped control RNAs (30). However, SL1 trans-splicing may play a role in nematode translation by adding the spliced leader at a relatively conserved distance from the AUG of the recipient RNA endogenous open reading frame that is optimal for translation of TMG-capped RNAs (30). Overall, however, the function of trans-splicing in several organisms remains unknown, and it remains to be determined what other functions trans-splicing may serve.

The spliced leader sequence varies among different trans-splicing organisms. However, within a group of trans-splicing organisms, the SL sequence typically exhibits high sequence conservation. For example, the 22-nucleotide SL1 spliced leader is almost absolutely conserved in all nematodes. The high sequence conservation of the nematode SL may be required as the sequence constitutes part of the SL RNA promoter in Ascaris and contributes to the ability to translate mRNAs with the trimethylguanosine cap (30, 31). Trans-splicing has been identified in four major and divergent flatworm groups, triclads, polyclads, trematodes (parasitic flukes), and cestodes (tapeworms) (8, 13–15, 20, 32). Flatworm spliced leaders exhibit a high degree of length and sequence variation, more than observed in some groups of trans-splicing organisms. The SL sequences range from 34 to 52 nucleotides in length (52 nucleotides is the longest SL known) and often have less than 50% nucleotide identity. The length and sequence variation in the SL is also accompanied by some variation in predicted flatworm SL RNA structure. Despite the significant SL length and sequence variation, several phylogenetically conserved nucleotides are present in divergent flatworm spliced leaders. Among these are an invariant 3'-terminal AUG. This is a unique characteristic of the flatworm SL not seen in spliced leaders from other organisms. In our original studies on schistosomes, the presence of the 3'-terminal SL AUG led us ask whether a function of transsplicing in flatworms might be to provide recipient mRNAs with an initiator methionine (33). Our initial sequence characterization of a small set of trans-spliced schistosome RNAs indicated that the majority of trans-spliced mRNAs contained an independent and endogenous 3' initiator methionine for the primary open reading frame of the recipient mRNA. For these mRNAs, the SL AUG was upstream and out-of-frame with the primary open reading frame of the mRNA. Interestingly, we also identified two mRNAs for which the SL AUG was in-frame with the primary open reading frame of the mRNA. However, no additional bioinformatic or functional data were available to support the use of the SL AUG as an initiator methionine.

Recently, large scale EST and cDNA sequencing efforts have been conducted on schistosome mRNAs (34, 35). In addition, during the last decade, genomic, cDNA, and EST sequencing efforts have increased enormously the number of predicted and known protein sequences. These new sequence data have enabled us to further examine the question of whether the flatworm SL AUG serves to provide recipient mRNAs with a required initiator methionine for expression of the endogenous mRNA open reading frame. In the current study, we have provided extensive bioinformatic data demonstrating that the flatworm spliced leader contributes an initiator AUG to some mRNAs necessary for the synthesis of N-terminal residues conserved in the orthologous proteins of other organisms. In addition, we developed and used methods for transient RNA transfection to demonstrate that the schistosome-spliced leader AUG can functionally serve as an initiator methionine in vivo. Our results have expanded the known functions of trans-splicing and demonstrate that spliced leader addition in flatworms serves to contribute an initiator methionine for expression of some recipient mRNA open reading frames.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bioinformatic Analyses—Schistosome cDNAs with the spliced leader sequence were identified in the NCBI non-redundant and EST databases as well as the Schistosoma mansoni GeneDB using BLASTN. Spliced leader cDNAs were screened to eliminate clones containing non-terminal SL sequences. Non-terminal SL sequences can represent as high as 20–25% of the clones.

5'-terminal SL clones were analyzed to determine whether the SL AUG was in-frame with the major open reading frame (ORF) of the mRNA using either the NCBI ORF Finder or MacVector Software (Accelrys, San Diego, CA). cDNAs with two approximately equivalent ORFs were eliminated from the analysis, and only cDNAs with ORFs of at least 150 nucleotides were characterized. Clones with the SL AUG in-frame with the major mRNA ORF were then analyzed by BLASTX against protein databases. Resulting pairwise alignments derived from BLASTX were initially analyzed to determine whether 1) the SL AUG-initiated protein had similarity with proteins in the data base between the SL AUG and the next downstream AUG and 2) if the similarity in these residues was phylogenetically conserved among orthologous proteins. In many cases, the orthologous proteins were extracted from the databases and subjected to multiple sequence alignment with the predicted schistosome proteins to confirm conservation of residues. To be considered a cDNA that uses the SL AUG to initiate open reading frames, a minimum of 5 identical or functionally similar and phylogenetically conserved residues must be present between the SL AUG and the downstream AUG. The majority of SL AUG-initiated open reading frames exhibit much higher similarity (a higher percentage of identity and similarity between the SL AUG and next downstream AUG) with orthologous proteins. In addition, most assigned SL AUG-initiated proteins are also present in diverse phylogenetic groups representing vertebrates and at least two invertebrate groups.

RNA Preparation for Transfection—PCR-generated templates for in vitro transcription were synthesized from pRL-null (Renilla luciferase) or pGL3 (firefly luciferase)(Promega, Madison, WI) using primers that introduce a T7 promoter and 5'-UTR sequences at the 5' end of the luciferase ORF and a 60- or 85-nucleotide poly-A tail at the 3' end. The in vitro transcription reactions (T7 Megascript, Ambion, Austin, TX) typically contained 1 µg of template/20-µl reaction, 10 mM ATP, 10 mM CTP, 10 mM UTP, 12 mM cap analogue (m7GpppG or m 2,2,73GpppG), 1.5 mM GTP, 1x reaction buffer, and 1x T7 polymerase mix and were carried out at 37 °C for 4 h. After DNase I treatment, the mRNA transcripts were extracted with TRIzol (Invitrogen), and the RNAs were precipitated twice, once with isopropyl alcohol and then with 0.5 M ammonium acetate/ethyl alcohol. Precipitated RNAs were further washed with 70% ethyl alcohol, dissolved in water, quantitated spectrophotometrically, and examined by agarose-formaldehyde denaturing gel electrophoresis. Analysis of cap orientation on transcripts was carried out as described (36) and indicated that greater than 80% of the caps were added in the correct orientation.

Schistosome Materials—Mice and hamsters infected with S. mansoni were provided by an National Institutes of Health contract through the Biomedical Research Institute and Fred Lewis. Additional S. mansoni-infected mice and hamsters were generously provided by Edward Pearce and Philip LoVerde, respectively. Adult schistosomes were perfused from the hepatic portal vasculature and maintained in RPMI 1640 with 10% FCS and 200 µg/ml penicillin/streptomycin at 37 °C and 5% CO2. Eggs were isolated from livers by gentle homogenization of infected livers in 1.5% saline followed by sequential filtering of the eggs through screens as described previously (37, 38). Purified eggs were suspended in spring or MilliQ water to stimulate hatching of the miracidial larval stage. The larval stages were collected by migration toward light and concentrated by gentle centrifugation at 4 °C (38). Miracidia were either used directly for biolistic experiments or transformed into sporocysts by 18–24 h of incubation in MEMSE-J with 1% bovine serum albumin and 200 µg/ml gentamycin at 26 °C in 5% CO2 and O2 (39, 40). Following 18–24 h of incubation, the transformed sporocysts were maintained at 26 °C in MEMSE-J with 5% FCS and 200 µg/ml penicillin/streptomycin in 5% CO2 and O2.

RNA Biolistics—Preparation of gold microcarriers and biolistics were performed essentially as described previously (41) with the following modifications: 1) gold particles were spherical ~2.2 µm gold from Degussa (10KM)(dmc2 Metals Group, South Plainfield, NJ) and 2) instead of lyophilization of the RNA onto the gold carriers, an alcohol precipitation was used. In vitro transcribed RNAs were precipitated onto gold particles using 2.5 M ammonium acetate/ethyl alcohol precipitation using either 0.25–5 µg of Renilla luciferase reporter RNA or 0.25–5 µg of firefly luciferase RNA/1 mg of gold particles. Luciferase activity in worms is RNA dose-dependent. The RNA/gold pellet was washed with 200 µl of ice-cold ethyl alcohol, resuspended in ice-cold 100% ethyl alcohol (18 µl/mg gold), and spread onto macrocarriers and processed as described previously (41). Binding of RNA onto the gold particles was evaluated by formaldehyde agarose gel analysis to determine the integrity of RNA bound to the beads, and RiboGreen (Molecular Probes) (42) was used to quantitate RNA loaded onto the particles for data normalization. Analysis of RNA bound to beads indicated that little to no degradation of the RNA occurred prior to particle bombardment and that typically 80–85% of the precipitated RNA was bound onto the gold particles.


Figure 1
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1.
Conservation of flatworm spliced leader 3'-terminal AUG. A, multiple sequence alignment of flatworm spliced leaders (8, 13–15, 20, 32). B, consensus sequences for schistosome translation initiation (non-SL = non-trans-spliced mRNAs) and the 3' ends of flatworm spliced leaders. The translation initiation consensus was determined as described (33) on 300 characterized schistosome mRNAs. The length and 3' ends of the flatworm SLs are illustrated. Dots within the consensus region indicate identical nucleotides (nts). The dashes preceding the consensus represent upstream sequence that is variable in the spliced leaders. C, nucleotide frequencies at positions –1 to –3 for 300 non-trans-spliced schistosome cDNAs.

 
Freshly perfused adult schistosomes were incubated in medium in 5% CO2 at 37 °C for 3 h to overnight prior to bombardment. 30 worm pairs in medium were carefully placed into the center of 35-mm Petri dishes, and just prior to bombardment, the medium was removed. The adults were bombarded with 1 mg of RNA-coated gold particles in a Bio-Rad Biolistic PDS-1000/HE particle delivery system at 15 inches of mercury of chamber vacuum, target distance of 3 cm (stage 1), and 1,550-psi particle acceleration pressure. In many experiments, the adults were immediately bombarded a second time. This effectively doubles the level of expression observed. After bombardment, 2–3 ml of RPMI plus 10% FCS and 200 µg/ml penicillin/streptomycin was added, and the adults were incubated at 37 °C in 5% CO2 for 3 h before the adults were collected, washed in phosphate-buffered saline, pelleted, and frozen prior to lysis for luciferase assays. Evaluation of gold particle delivery was carried out as described previously (41).

For particle bombardment of miracidia and sporocyst, freshly isolated miracidia or 18–24-h transformed sporocysts were suspended in MEMSE-J with 1% bovine serum albumin and 200 µg/ml gentamycin, and ~1000 miracidia or sporocysts were gently spread onto the center of 35-mm Petri dishes in a minimal volume. Optimal biolistics parameters were determined to be 15 inches of mercury of chamber vacuum, target distance of 6 cm (stage 2), and 450-psi particle acceleration pressure. Following bombardment, 2 ml of Media F or MEMSE-J plus 1% bovine serum albumin or 5% FCS were added, and the miracidia or sporocysts were incubated at 26 °C in 5% CO2 and O2 for 3 h before they were collected, washed in media, pelleted, and frozen prior to lysis for luciferase assays. Bombarded miracidia develop into viable sporocysts that can be maintained for several weeks.

RNA Electroporation of Sporocysts—600 –2000, 18–24-h-old sporocysts were resuspended in 100 µl of MEMSE-J with 1% FCS. The sporocysts were placed into prechilled 0.4-cm electroporation cuvettes, RNA was added to 50 µg/ml, and electroporation was carried out in a BTX ECM 830 (square wave) electroporator (BTX, Holliston, MA) at 280 volts using a 0.5-ms pulse length. Following electroporation, the sporocysts were suspended in 2 ml of MEMSE-J with 5% FCS/200 µg/ml penicillin/streptomycin and incubated at 26 °C in 5% CO2 and O2 for3h before they were collected, washed in media, pelleted, and frozen prior to lysis for luciferase assays.

Luciferase and Protein Assays—Frozen pellets of adult or larval schistosome stages were resuspended in 150 µl of the recommend lysis buffer (Promega, Madison, WI) and homogenized in Kontes disposable PEL-LET PESTLES® with Microtubes (Vineland, NJ) several times using several freeze-thaw cycles and multiple passes with a motor-driven pestle. The lysates were cleared by centrifugation, and aliquots (20 µl) of the supernatant were assayed using the Promega Renilla, luciferase, or Dual-Luciferase assay systems with a Sirius luminometer (Zylux Corp., Oak Ridge, IN)) (41). Dual-Luciferase assays were used in conjunction with co-transfection of two reporters (firefly and Renilla luciferase) as normalization for transfection efficiency and biological variation. Protein assays on lysates were carried out using the Pierce BCA protein assay kit and Compat-Able protein assay preparation reagent set (Pierce). Experimental data were similar with or without normalization to a co-transfected reporter and to protein concentrations in sample lysates. Thus, some experimental data are presented with normalization, whereas other data are not.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conservation of the Flatworm SL AUG and Its Sequence Context—An alignment of the known flatworm spliced leaders and a subalignment of their 3' ends are presented in Fig. 1, A and B. The alignments illustrate the absolute sequence conservation of the 3'-terminal AUG of flatworm spliced leaders as well as the AUG sequence context. We previously derived a schistosome translation initiation AUG consensus from a small set of non-trans-spliced schistosome mRNAs. Re-evaluation of this consensus in light of additional sequences (~300) indicates that it is still valid with the larger sampling (33) (Fig. 1C). The non-trans-spliced mRNA translation initiation consensus along with the consensus of the flatworm SL AUG are shown in Fig. 1B. The schistosome translation initiation AUG context for non-trans-spliced mRNAs conforms to that observed in higher eukaryotes and a variety of invertebrates (43–45) with a highly conserved purine in the –3 position (Fig. 1C). Notably, all the flatworm SL AUGs have a U in the –3 position instead of a purine.

Bioinformatic Analysis of SL cDNAs and ESTs—We previously noted that the schistosome SL AUG was typically not in-frame with major open reading frames of recipient mRNAs (33). However, we observed two mRNAs (corresponding to ~5% of the trans-spliced mRNAs we originally identified) for which the SL AUG was in-frame with the major ORF of the recipient mRNA. Available bioinformatic data at the time did not support the conservation of the protein sequence between SL AUG and the next 3'-AUG with orthologous protein sequences in databases. The large increase in known and predicted protein sequences from a breadth of organisms, as well new EST and cDNA data for schistosomes (34, 35), led us to further examine whether we could derive additional data to support the use of the conserved flatworm spliced leader AUG as an initiator methionine. We re-examined trans-spliced mRNAs we previously described and identified a large number of new trans-spliced schistosome mRNAs in both EST and nucleotide databases (~250,000 ESTs and cDNAs) (34, 35). These mRNA sequences were derived from two different schistosome species, S. mansoni and Schistosoma japonicum, which have identical SL sequences. Only a small percentage of the known cDNAs and EST sequences contains the schistosome SL. For example, of the ~150,000 known S. mansoni EST sequences, ~245 (0.16%) clones contained the spliced leader, whereas of the ~97,000 S. japonicum EST sequences, ~845 (0.9%) have the spliced leader. These values are an under-representation of the frequency of mRNAs with an SL for several reasons. First, unless specialized methods are used, full-length cDNAs are typically under-represented in libraries. Second, many of the S. mansoni ESTs represent sampling of internal cDNA fragments and cDNA synthesis, and sampling did not emphasize preparation or analysis of full-length clones (34). Recently, ~9,500 S. japonicum cDNAs sequences were deposited into the nucleotide data base. Of these ~9,500 cDNAs, 216, or 2.3% of the clones, have a 5'-terminal spliced leader (270 clones have the SL, but 54 of these clones exhibited the SL in a non-terminal position). For our analyses, we examined all available cDNAs with a 5'-terminal SL in current databases (NCBI nucleotide and dbEST and S. mansoni GeneDB) to identify clones that might use the SL AUG as an initiator methionine. However, the 216 S. japonicum SL cDNAs derived from the newly deposited 9,500 cDNAs were used to calculate the percentages of clones with the SL AUG in-frame and other frequencies as these are primarily full-length and better characterized mRNAs.

The SL sequence is predominantly out-of-frame with the AUG of the major open reading frame for the majority of the SL mRNAs (72%). 28% of the cDNAs and ESTs contain the SL AUG in-frame with the main open reading frame. Many mRNAs often use the first in-frame AUG as the initiator methionine, particularly when present in an appropriate sequence context. However, there are a growing number of examples in which the first AUG may not be used as the initiation codon (46–48). In addition, as little is known regarding translation initiation in flatworms, it is possible that trans-spliced mRNAs with the potentially poor sequence context of the SL AUG (it lacks a purine at –3 when compared with non-trans-spliced schistosome mRNAs and other invertebrates) might preferentially initiate translation from a downstream AUG in a better context.

N-terminal sequence for proteins derived from trans-spliced mRNAs containing the SL AUG in-frame with major ORF would provide definitive data regarding the initiator methionine of these proteins. However, these data are exceptionally difficult and expensive to obtain, particularly in parasitic organisms for which limited material is available. In the absence of these data, we chose to examine predicted SL AUG-initiated proteins for conserved amino acid residues between the SL AUG and the next 3'-AUG in orthologous proteins. If a predicted SL AUG-initiated protein contained conserved residues in orthologous proteins between the SL AUG and the next downstream AUG, these data would provide strong support that the SL AUG would be required for translation to produce a protein with N-terminal residues that are phylogenetically conserved for that particular protein. Of those cDNA clones that have the SL AUG in-frame with the major ORF (SL AUG in-frame clones), 35% have an in-frame Met within ~10 amino acids 3' of the SL AUG (10% of all SL clones identified). None of these cDNAs passed the similarity criterion described above for the amino acids between the SL AUG and the next in-frame AUG. These cDNAs may use the SL AUG (see below), or in some cases, the downstream AUG in these mRNAs might be used as the translation initiator methionine via a leaky scanning mechanism (49, 50). Of the remaining SL AUG in-frame clones, ~40% exhibited phylogenetically conserved sequence similarity between the SL AUG and the downstream methionine, supporting the use of the SL AUG as an initiator methionine. Thus, over 40 schistosome mRNAs representing ~10% of the total SL-containing clones were identified in the different databases that meet our criterion (see supplemental Table 1). Alignments illustrating the phylogenetic conservation of SL AUG-initiated N-terminal residues are shown in Fig. 2. These alignments are representative of the types of matches obtained and are presented to illustrate the evidence for conservation of N-terminal residues. Most alignments for predicted proteins initiated by the SL are strongly supportive of the SL AUG to generate a functional protein based on phylogenetic conservation of residues in orthologous proteins (Fig. 2). However, some alignments (~15%) are not as supportive as those shown. The majority of the predicted schistosome proteins are similar to known proteins with a discrete function. Only a small percentage of the proteins is similar to unknown, hypothetical proteins. This suggests that the use of the SL AUG for translation initiation is not generally a property of schistosome-specific proteins. Overall, these data provided strong support that a number of trans-spliced schistosome mRNAs would require the SL AUG as an initiator methionine to produce proteins with the requisite phylogenetically conserved N-terminal residues.

Luciferase RNA Reporter Transfection—We developed methods for RNA transfection using biolistics and electroporation into several stages of the schistosome life cycle, including miracidia (a free-swimming stage infecting the freshwater snail intermediate host), sporocysts (derived from miracidia that undergo polyembryony within the intermediate snail host), and adults (the sexual form in the vertebrate host). Following optimization of parameters for both biolistic and electroporation methods (see "Materials and Methods" for conditions used), we prepared luciferase (firefly or Renilla) RNAs to evaluate whether the schistosome SL AUG could be used to produce functional luciferase protein in vivo as measured by assay for luciferase activity. The initial test RNAs synthesized are illustrated in Fig. 3A. Wild-type firefly or Renilla luciferase RNAs were used as controls for transfection and translation. The firefly RNA control (firefly) consisted of an m7GpppG-capped 36-nucleotide 5'-UTR, firefly luciferase ORF, 50-nucleotide SV40 3'-UTR, and a 3' 60-nucleotide poly(A)-tail. The control Renilla RNA (Renilla) consisted of an m7GpppG-capped 32-nucleotide 5'-UTR, Renilla ORF, 50-nucleotide SV-40 3'-UTR, and 3' 85-nucleotide poly(A)-tail. To test whether the spliced leader AUG could initiate translation of luciferase protein, the entire 5'-UTRs of these RNAs were substituted with the 36-nucleotide schistosome SL, placing the SL AUG as the initiator methionine in-frame with the Renilla or firefly luciferase ORF (firefly or Renilla S. mansoni SL AUG). Test RNAs were introduced into schistosomes either by biolistics (adults, miracidia, and sporocysts) or electroporation (sporocysts). 3 h following RNA introduction, the samples were collected, frozen, lysed, and assayed for luciferase activity. As illustrated in Fig. 3B, introduction of the control firefly luciferase RNAs into different schistosome stages led to significant levels of luciferase activity in parasite lysates 3 h after RNA introduction. Replacement of the positive control RNA 5'-UTR with the schistosome-spliced leader such that the SL AUG replaced the luciferase AUG also resulted in luciferase activity in the parasite lysates. These data suggested that the schistosome-spliced leader AUG can function as a translation initiator methionine. To further test whether the luciferase activity observed was dependent on the SL AUG as the initiator methionine, we synthesized RNAs with the SL AUG mutated to GCG (firefly S. mansoni SL AUG to GCG and Renilla S. mansoni SL AUG to GCG). Mutation of either the wild-type luciferase or schistosome-spliced leader initiator AUG to GCG (AUG to GCG mRNAs) resulted in almost complete loss of luciferase activity in schistosomes (Fig. 3, B–D). These data are consistent with previous experiments demonstrating that the N-terminal 8 amino acids of firefly luciferase are required for luciferase activity (51). Data illustrated in Fig. 3, C and D, were normalized for both transfection efficiency (co-transfection with a second RNA reporter) and protein content in schistosome lysates. Thus, differences in luciferase activity were not a function of variation in transfection frequencies or schistosome material. These data demonstrated that the schistosome SL AUG can serve as an initiator methionine for translation.


Figure 2
View larger version (68K):
[in this window]
[in a new window]
  		FIGURE 2.
N-terminal residues of SL AUG-initiated proteins are conserved in orthologous proteins. Predicted proteins encoded by the SL AUG to the next in-frame AUG were aligned with orthologous proteins using the MacVector implementation of Clustal. Darkly shaded residues are identical amino acids, and lightly shaded residues are functionally similar. These are representative alignments for the proteins identified. A, schistosome accession number AY811984 [GenBank] , cyclophilin J (peptidylproly isomerase protein 3). B, schistosome accession number AY223260 [GenBank] , nucleotide-binding protein 2 (Mrp, ATPase involved in chromosome partitioning). C, schistosome accession number AY815507 [GenBank] , acyl CoA binding domain protein. D, schistosome accession number AY812760 [GenBank] , steroid, 3-oxo-5-{alpha}-steroid 4-dehydrogenase (steroid 5-{alpha}-reductase).

 


Figure 3
Figure 3
View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 3.
The schistosome SL AUG can serve as an initiator methionine for in vivo luciferase translation. A, illustration of 5' sequence of RNAs and initiator AUGs for firefly or Renilla RNA transfection. B, firefly luciferase activity following biolistic introduction of test RNAs into schistosome adults, miracidia, and sporocysts. Test RNAs were introduced into different schistosome life cycle stages, the schistosomes were incubated for 3 h for translation of the RNA, the schistosomes were collected and lysates were prepared, and the lysates were assayed for luciferase activity. C, normalized firefly luciferase activities in co-transfected, electroporated sporocysts. The illustrated firefly luciferase RNAs were electroporated along with a second Renilla RNA as a transfection control into schistosome sporocysts (see Renilla in A). Luciferase activity in sporocysts was determined 3 h after transfection using the Dual-Luciferase assay. Firefly luciferase activity is illustrated normalized to both Renilla luciferase activity and protein concentration in sporocyst lysates. D, normalized Renilla luciferase activities in firefly co-transfected, electroporated sporocysts. Luciferase activity in sporocysts was determined 3 h after transfection using the Dual-Luciferase assay. Renilla luciferase activity is illustrated normalized to co-transfected firefly luciferase RNA activity and protein concentration in sporocyst lysates. These are representative experiments illustrating the mean ± S.E. with three replicates for each RNA sample evaluated.

 
Spliced Leader AUG Context and Translation—The SL AUG initiation context contains a U at –3. This is often considered a poor initiation context for some organisms. In a sampling of 300 non-trans-spliced schistosome cDNAs, U was present at –3 18% of the time (Fig. 1C). To further examine the SL AUG initiation context and the contribution of the spliced leader sequence to translation initiation efficiency, we compared the AUG initiation context as a function of the nucleotide at the –3 position and in the context of the spliced leader sequence. As illustrated in Fig. 4, substitution of the SL U at the –3 position to A led to significant increases in translation efficiency (compare SL UGC AUG to SL AGC AUG) for both firefly and Renilla reporters with the spliced leader. An A at the –3 position is the preferred nucleotide present 54% of the time in non-trans-spliced schistosome cDNAs (Fig. 1C). Thus, the increases in translation efficiency with A at –3 are consistent with the apparent preferred AUG context in non-trans-spliced schistosome cDNAs (Fig. 4).

As the UGC of the SL AGU is apparently a suboptimal context for initiation (when compared with AGC), we examined whether the upstream SL sequence contributed to the translation context by comparing translation of the SL UGC AUG with the first 30 nucleotides of the SL substituted with random nucleotides (N30). Notably, the translation efficiency of the N30 was as efficient as the SL in the 5'-UTR of the reporter RNA. These data indicated that the SL sequence itself does not contribute significantly to the use of the UGC AUG context. This may not be unexpected as 18% of schistosome cDNAs have a U at –3, and at least for the firefly luciferase RNA, mRNAs with UGC AUG are relatively efficiently translated (Fig. 4A). Overall, these data indicated that although a purine at –3 may be optimal in some contexts, mRNAs with a U at –3 can be relatively efficiently translated.

TMG Cap and in Vivo Translation—The addition of the flatworm spliced leader to the mRNA also results in the addition of an m 2,2,73GpppA cap to the RNA (8). Substitution of an m 2,2,73GpppG for the m7GpppG cap on the Renilla control RNA led to a reduction in translation when compared with the m7GpppG capped Renilla control RNA (Fig. 5). The addition of the TMG cap in the context of the SL did not improve translation of TMG-capped transcripts. Overall, reporter RNAs with the TMG and spliced leader were translated less efficiently than the m7G-capped control RNA.

SL AUG to Endogenous ORF AUG Distance—The addition of the spliced leader to recipient mRNAs that do not use the SL AUG as an initiator methionine places an AUG upstream from the major ORF AUG. In other organisms, this is known to be a potentially confounding factor that might require either leaky scanning or, if a short ORF is present, reinitiation mechanisms to facilitate initiation at the major ORF (44, 49, 50). To determine whether the flatworm SL might be added at a conserved distance from the endogenous ORF AUG, we examined 160 S. japonicum mRNAs to define the distance between the SL and ORF of endogenous AUG-initiated mRNAs. As shown in Fig. 6A, 50% of the clones acquire the spliced leader within 30 nucleotides of the major open reading frame AUG. The distribution within the 30 nucleotides is further illustrated in Fig. 6B.

Effect of an Upstream SL AUG on Downstream AUG Translation—To evaluate the effect of the upstream and out-of-frame SL AUG on translation of a downstream open reading frame, we selected the 5'-UTRs of two native mRNAs for analysis, one encoding a high voltage-activated calcium channel beta subunit 2 and another encoding a U2 small nuclear ribonucleoprotein B (Fig. 7A). The distance between the SL AUG and the open reading frame AUG for these mRNAs is 13 and 16 nucleotides, respectively. These distances fall within the typical range of the SL AUG to the ORF AUG as illustrated in Fig. 6. The translation efficiency of reporter RNAs with these 5'-UTRs introduced into sporocysts was compared with the wild-type control firefly 5'-UTR and the SL AUG 5'-UTR. Notably, as illustrated in the Fig. 7B, the upstream and out-of-frame SL AUG did not have a negative effect on firefly reporter translation. In fact, the 5'-UTRs derived from SL AUG out-of-frame mRNAs led to higher levels of luciferase activity in schistosome lysates than either the wild-type or SL AUG translation-initiated reporter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data have demonstrated that the spliced leader AUG is used as an initiator methionine for the open reading frames of some recipient schistosome mRNAs. This conclusion has been supported by protein similarity data that require the SL 3'-AUG as an initiator methionine to initiate synthesis of a protein with conserved N-terminal residues characteristic of orthologous proteins. In addition, RNA transfection experiments demonstrated that the schistosome SL AUG can be used as an initiator methionine for translation of test luciferase mRNAs.


Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 4.
Analysis of the schistosome AUG initiation context. A, firefly luciferase RNA reporter AUG initiation context. Sporocysts were electroporated with the illustrated firefly RNAs and a co-transfected Renilla luciferase RNA control. Luciferase activity was measured in sporocyst lysates 3 h after transfection using the Dual-Luciferase assay. Data were normalized to the co-transfected Renilla reporter and protein concentrations in the lysates. The firefly-S. man SL UGC AUG RNA (firefly S. mansoni SL AUG) and firefly-ACC AUG RNA (firefly) are illustrated in Fig. 3A. The firefly-S. man SL AGC AUG differs only in the substitution of an A at the –3 position, whereas the firefly-UGC AUG differs by substitution of UG at positions –2 and –3. Firefly-N30 UGC AUG contains the last 6 nucleotides of the spliced leader (UGC AUG) with the first 30 nucleotides of the spliced leader substituted with random nucleotides. B, Renilla luciferase RNA reporter AUG initiation context. Sporocysts were electroporated with the illustrated Renilla RNAs, and Renilla luciferase activity was measured 3 h after transfection. RNA designations are as in A except that they are Renilla RNAs. These are representative experiments illustrating the mean ± S.E. with three replicates for each RNA sample evaluated.

 
Bioinformatic Analyses of SL AUG-initiated Proteins—28% of the schistosome SL cDNA clones analyzed have the SL AUG in-frame with the major open reading frame of the recipient mRNA. Approximately ~38% of these clones have a 2nd in-frame Met within ~10 amino acids of the SL AUG. Some or many of these clones could use the SL AUG as the initiator methionine since the RNA transfection data demonstrated that the SL AUG can act as an initiator. In addition, in some contexts, these adjacent AUG codons have been shown to increase translation, and it remains to be determined whether this is true in schistosomes (52). Using relatively strict bioinformatic criteria requiring phylogenetically conserved sequence similarity between the residues encoded between the SL AUG and the next in-frame downstream AUG, we have identified a set of ~40 mRNAs that use the SL AUG. This is likely an underestimate, and a number of the mRNAs that do not pass these criteria may actually use the SL AUG as an initiator methionine. Analysis of the types of proteins encoded by clones with the SL AUG in-frame did not reveal any common patterns in types of proteins encoded.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5.
Translation of TMG-SL RNAs in schistosomes. A, sporocysts. The illustrated test Renilla RNAs were electroporated with a co-transfected Firefly luciferase RNA into sporocysts and luciferase activity measured 3 h after transfection using the Dual-Luciferase assay. Data were normalized to the co-transfected firefly reporter and protein concentrations in the sporocyst lysates. B, miracidia. The illustrated test Renilla RNAs were biolistically introduced into miracidia, and Renilla luciferase activity was measured 3 h after transfection. The RNAs are as illustrated in Fig. 3A except for the substitution of a TMG cap on some RNAs. These are representative experiments illustrating the mean ± S.E. with three replicates for each RNA sample evaluated. m7G, m7GpppG cap; TMG, = m 2,2,73GpppG cap.

 
SL AUG Initiation Context—Several features are known to influence the efficiency and/or fidelity of translation initiation at an AUG codon in vertebrates including 1) the mRNA cap, 2) the length of the 5'-UTR, 3) the context of the AUG codon, 4) the secondary structure 5' and 3' of the AUG, and 5) the presence of upstream AUGs or ORFs (49, 50). The levels of SL AUG-derived firefly luciferase activity were on average 50% higher than the control 5'-UTR for firefly luciferase. However, in contrast, Renilla luciferase activity with the SL AUG 5'-UTR was typically ~30% less than the control Renilla 5'-UTR (data not shown). Our interpretation of these data is that the SL AUG provides a reasonably efficient translation initiation context. However, a better understanding of the comparative efficiency of the 5'-UTRs of trans-spliced versus non-trans-spliced mRNAs will require additional analyses using several different non-trans-spliced, schistosome mRNA 5'-UTRs.


Figure 6
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 6.
Spliced leader AUG to predicted main open reading frame initiator methionine distance. A set of 160 trans-spliced S. japonicum cDNAs that do not have the spliced leader AUG in-frame with the main open reading frame were analyzed for the nucleotide distance between the SL AUG and the initiator methionine of the ORF. The percentage of clones was plotted against the nucleotide distance. A, distance in 10-nucleotide intervals to 100 nucleotides, distance in 50-nucleotide intervals from 101 to 200, and distance greater than 200 nucleotides are illustrated. B, distance in 5-nucleotide intervals from 1 to 30 nucleotides is illustrated.

 
The schistosome SL AUG initiation context lacks the purine at the –3 position (a U is present) that is typical of initiation contexts for non-trans-spliced schistosome mRNAs, other invertebrates, or the general vertebrate Kozak consensus (GCCA/GCCAUGG) (43, 45, 49, 50). Analysis of non-trans-spliced schistosome AUG contexts indicated that ~18% of the mRNAs have a U at this position. The most common nucleotide at –3 is an A present 54% of the time followed by a G at 21%. The control luciferase RNAs used in this study have a Kozak consensus (ACC) for –1 to –3. Substituting UGC (the SL AUG –1 to –3) for the Kozak context sequence in the control RNA led to only a small drop in translation efficiency (Fig. 4A). Furthermore, efficient use of the SL AUG context (UGC) is not dependent on the upstream spliced leader sequence as a 5'-UTR with 30 random 5'-nucleotides (N30 UGC AUG) substituted for the upstream SL sequence does not have a large effect on translation. However, substitution of an A for U at the –3 position leads to a significant increase in overall luciferase activity (Fig. 4). Thus, the relatively high frequency of A at –3 observed in cDNAs is reflective of a more optimal translation initiation context. Overall, these data indicated that aspects of the translation initiation context in schistosomes may be somewhat flexible and are complex.


Figure 7
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 7.
Effect of upstream and out-of-frame SL AUG on luciferase activity. A, an illustration of the test RNA 5'-UTRs examined. The underlined sequence represents the spliced leader sequence, and the bold ATG is the initiator methionine for the main open reading frame. B, luciferase activity of a firefly reporter RNAs as a function of the native, trans-spliced upstream and out-of-frame SL AUG 5'-UTR. The illustrated reporter RNAs were electroporated along with a co-transfected Renilla RNA reporter into sporocysts. Luciferase activity 3 h after transfection was measured using a Dual-Luciferase assay, and the firefly activity was normalized to both Renilla luciferase and protein concentration. This is a representative experiment illustrating the mean ± S.E. with three replicates for each RNA sample evaluated.

 
The addition of SL AUG Out-of-Frame—The majority of trans-spliced flatworm mRNAs do not have the spliced leader AUG in-frame with the major open reading frame. As illustrated in Fig. 6, the spliced leader is typically trans-spliced within 30 nucleotides (54% of the mRNAs) of the initiator methionine of the recipient mRNA. No general patterns of encoded proteins were observed for these mRNAs as they appear to represent a diversity of protein types in a variety of cellular pathways. This is also true in the tapeworm Echinococcus (53).

The addition of an upstream and out-of-frame SL derived AUG to the 5'-UTR of the recipient mRNAs might require leaky scanning or reinitiation mechanisms for translation of these mRNAs. For some oncogenes and growth factors, an upstream and out-of-frame AUG can lead to low or regulated translation of these mRNAs (44). Addition of the native 5'-UTRs of two trans-spliced mRNAs with the SL AUG upstream and out-of-frame with the reporter AUG did not result in a reduction of overall luciferase activity. In fact, an increase in translation over both the wild-type and the SL AUG 5'-UTR was observed (Fig. 7B). The test mRNAs have 13 or 16 nucleotides between the SL AUG and the reporter AUG. This distance is within the range observed for the majority of trans-spliced mRNAs that have the SL AUG out-of-frame with the ORF. The addition of the SL AUG less than 30 nucleotides from the ORF AUG might be optimal to minimize deleterious effects of an upstream SL AUG. Thus, the flatworm translation machinery may have evolved to accommodate the upstream AUG within a specified distance. However, additional experiments are required to rigorously examine this, and it remains to be determined whether shorter or longer distances between the SL AUG and the ORF initiator methionine have differential effects on translation initiation.

The nematode SL also typically splices close to the recipient mRNA initiator methionine. We recently demonstrated that this spacing is optimal for translation in vitro (30). However, the nematode SL does not contain a 3'-terminal AUG. We previously proposed that heterogeneous transcription initiation from trans-spliced genes may require that trans-splicing trim heterogeneous 5'-UTRs to generate uniform 5'-UTRs with an appropriate translation initiation context lacking additional upstream AUGs (30). The spacing between the SL and initiator methionine in both flatworms and nematodes is consistent with this hypothesis. However, although transcription of Ascaris trans-spliced genes appears heterogeneous, which is consistent with this hypothesis, it has been difficult to provide definitive data on the heterogeneous transcription sites (30).

TMG Cap and Translation—The addition of the TMG cap leads to a reduction in translation of test mRNAs in schistosomes even when this cap is present on the SL (Fig. 5). This is similar to what we observed for TMG-capped reporters in nematode embryos (30). Thus, under the experimental conditions evaluated, translation of TMG-capped RNAs in both flatworms and nematodes is in general less efficient than m7G-capped RNAs.

Translation Context and RNA Stability—In the current studies, we have not directly examined the mRNA half-lives for various test mRNAs. The large 50–100-fold differences in luciferase activity observed when the SL AUG is mutated to GCG are not likely explained by differences in RNA stability. Other RNAs examined with 1–2-nucleotide substitutions are also not likely to have significant differences in RNA stability. In addition, in separate studies in nematodes, the SL sequence and random sequence substitutions in the 5'-UTR did not have a significant effect on mRNA stability of the test RNAs (30).3 Overall, although we think that the current data are reflective of primarily translation initiation efficiency, some differences in mRNA half-life may also contribute to the overall levels of translation we have measured.

Conclusions—SL trans-splicing was also recently described in cestodes, a distinct flatworm group commonly known as tapeworms (13, 32). Analysis of a small set of trans-spliced mRNAs for two genera indicated that several of the trans-spliced mRNAs were likely to use the SL AUG based on the conserved N-terminal residues criterion applied here. A subsequent study also identified a high percentage of SL cDNA clones that were likely to use the SL AUG based on the SL AUG in-frame with an open reading frame of at least 99 nucleotides (53). Our bioinformatic and functional schistosome data provides additional support to suggest that the tapeworm SL AUG is also likely to serve as an initiator methionine for some mRNAs.

In summary, bioinformatic and transfection data demonstrated that the 3'-terminal AUG of the schistosome-spliced leader can be used as an initiator methionine. These findings have added a novel function to the list of functions for trans-splicing.


   FOOTNOTES
 
* This work was supported by grants from Ellison Medical Foundation (Grant ID-IA-0037) and the National Institutes of Health (Grant AI49558) and by UCHSC startup funds (to R. E. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental table showing schistosome mRNAs using the SL AUG as an initiator methionine. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Mail Stop 8101, P. O. Box 6511, 12801 East 17th Ave., Aurora, CO 80045. Tel.: 303-724-3226; Fax: 303-724-3215; E-mail: Richard.Davis{at}uchsc.edu.

2 The abbreviations used are: SL, spliced leader; TMG, trimethylguanosine; ORF, open reading frame; UTR, untranslated region; FCS, fetal calf serum; EST, expressed sequence tag. Back

3 L. S. Cohen, M. Mikhli, M. Jankowska, J. Stepinski, E. Darzynkiewicz, and R. E. Davis, submitted Back


   ACKNOWLEDGMENTS
 
We thank Phil LoVerde and Ed Pearce for generously providing protocols and schistosome-infected hamsters and mice and an anonymous reviewer for helpful suggestions to the manuscript. Thanks to Fred Lewis and the Biomedical Research Institute for providing schistosome-infected mice and hamsters and for valuable training, assistance, and support. We thank Anthony Esposito, Melissa Katz, and Nathan Netsanet for contributions to the identification and analysis of trans-spliced schistosome mRNAs. Particular thanks to Phil Newmark for sharing the planarian spliced leader sequences prior to publication and Edward Darzynkiewicz for cap analogs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hastings, K. E. (2005) Trends Genet. 21, 240–247[CrossRef][Medline] [Order article via Infotrieve]
  2. Nilsen, T. W. (2001) Trends Genet. 17, 678–680[CrossRef][Medline] [Order article via Infotrieve]
  3. Davis, R. E. (1996) Parasitol. Today 12, 33–40[CrossRef][Medline] [Order article via Infotrieve]
  4. Van Doren, K., and Hirsh, D. (1990) Mol. Cell. Biol. 10, 1769–1772[Abstract/Free Full Text]
  5. Maroney, P. A., Hannon, G. J., and Nilsen, T. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 709–713[Abstract/Free Full Text]
  6. Liou, R. F., and Blumenthal, T. (1990) Mol. Cell. Biol. 10, 1764–1768[Abstract/Free Full Text]
  7. Thomas, J. D., Conrad, R. C., and Blumenthal, T. (1988) Cell 54, 533–539[CrossRef][Medline] [Order article via Infotrieve]
  8. Rajkovic, A., Davis, R. E., Simonsen, J. N., and Rottman, F. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8879–8883[Abstract/Free Full Text]
  9. Stover, N. A., and Steele, R. E. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5693–5698[Abstract/Free Full Text]
  10. Pouchkina-Stantcheva, N. N., and Tunnacliffe, A. (2005) Mol. Biol. Evol. 22, 1482–1489[Abstract/Free Full Text]
  11. Ganot, P., Kallesoe, T., Reinhardt, R., Chourrout, D., and Thompson, E. M. (2004) Mol. Cell. Biol. 24, 7795–7805[Abstract/Free Full Text]
  12. Vandenberghe, A. E., Meedel, T. H., and Hastings, K. E. (2001) Genes Dev. 15, 294–303[Abstract/Free Full Text]
  13. Brehm, K., Jensen, K., and Frosch, M. (2000) J. Biol. Chem. 275, 38311–38318[Abstract/Free Full Text]
  14. Davis, R. E. (1997) Mol. Biochem. Parasitol. 87, 29–48[CrossRef][Medline] [Order article via Infotrieve]
  15. Davis, R. E., Singh, H., Botka, C., Hardwick, C., Ashraf el Meanawy, M., and Villanueva, J. (1994) J. Biol. Chem. 269, 20026–20030[Abstract/Free Full Text]
  16. Tessier, L. H., Keller, M., Chan, R. L., Fournier, R., Weil, J. H., and Imbault, P. (1991) EMBO J. 10, 2621–2625[Medline] [Order article via Infotrieve]
  17. Krause, M., and Hirsh, D. (1987) Cell 49, 753–761[CrossRef][Medline] [Order article via Infotrieve]
  18. Van der Ploeg, L., Liu, A., Michels, P., De Lange, T., Borst, P., Majumder, H., Weber, H., Veeneman, G., and Van Boom, J. (1982) Nucleic Acids Res. 10, 3591–3604[Abstract/Free Full Text]
  19. Boothroyd, J., and Cross, G. (1982) Gene (Amst.) 20, 279–287[CrossRef]
  20. Zayas, R. M., Bold, T. D., and Newmark, P. A. (2005) Mol. Biol. Evol. 22, 2048–2054[Abstract/Free Full Text]
  21. Blumenthal, T., and Gleason, K. S. (2003) Nat. Rev. Genet. 4, 110–118[CrossRef]
  22. Blumenthal, T., Evans, D., Link, C. D., Guffanti, A., Lawson, D., Thierry-Mieg, J., Thierry-Mieg, D., Chiu, W. L., Duke, K., Kiraly, M., and Kim, S. K. (2002) Nature 417, 851–854[CrossRef][Medline] [Order article via Infotrieve]
  23. Davis, R. E., and Hodgson, S. (1997) Mol. Biochem. Parasitol. 89, 25–39[CrossRef][Medline] [Order article via Infotrieve]
  24. Spieth, J., Brooke, G., Kuersten, S., Lea, K., and Blumenthal, T. (1993) Cell 73, 521–532[CrossRef][Medline] [Order article via Infotrieve]
  25. Muhich, M. L., and Boothroyd, J. C. (1988) Mol. Cell. Biol. 8, 3837–3846[Abstract/Free Full Text]
  26. Johnson, P. J., Kooter, J. M., and Borst, P. (1987) Cell 51, 273–281[CrossRef][Medline] [Order article via Infotrieve]
  27. Liang, X. H., Haritan, A., Uliel, S., and Michaeli, S. (2003) Eukaryot. Cell 2, 830–840[Free Full Text]
  28. Huang, X. Y., and Hirsh, D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8640–8644[Abstract/Free Full Text]
  29. Zorio, D. A., Cheng, N. N., Blumenthal, T., and Spieth, J. (1994) Nature 372, 270–272[CrossRef][Medline] [Order article via Infotrieve]
  30. Lall, S., Friedman, C. C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E., and Davis, R. E. (2004) J. Biol. Chem. 279, 45573–45585[Abstract/Free Full Text]
  31. Hannon, G. J., Maroney, P. A., Ayers, D. G., Shambaugh, J. D., and Nilsen, T. W. (1990) EMBO J. 9, 1915–1921[Medline] [Order article via Infotrieve]
  32. Brehm, K., Hubert, K., Sciutto, E., Garate, T., and Frosch, M. (2002) Mol. Biochem. Parasitol. 122, 105–110[CrossRef][Medline] [Order article via Infotrieve]
  33. Davis, R. E., Hardwick, C., Tavernier, P., Hodgson, S., and Singh, H. (1995) J. Biol. Chem. 270, 21813–21819[Abstract/Free Full Text]
  34. Verjovski-Almeida, S., DeMarco, R., Martins, E. A., Guimaraes, P. E., Ojopi, E. P., Paquola, A. C., Piazza, J. P., Nishiyama, M. Y., Jr., Kitajima, J. P., Adamson, R. E., Ashton, P. D., Bonaldo, M. F., Coulson, P. S., Dillon, G. P., Farias, L. P., Gregorio, S. P., Ho, P. L., Leite, R. A., Malaquias, L. C., Marques, R. C., Miyasato, P. A., Nascimento, A. L., Ohlweiler, F. P., Reis, E. M., Ribeiro, M. A., Sa, R. G., Stukart, G. C., Soares, M. B., Gargioni, C., Kawano, T., Rodrigues, V., Madeira, A. M., Wilson, R. A., Menck, C. F., Setubal, J. C., Leite, L. C., and Dias-Neto, E. (2003) Nat. Genet. 35, 148–157[CrossRef][Medline] [Order article via Infotrieve]
  35. Hu, W., Yan, Q., Shen, D. K., Liu, F., Zhu, Z. D., Song, H. D., Xu, X. R., Wang, Z. J., Rong, Y. P., Zeng, L. C., Wu, J., Zhang, X., Wang, J. J., Xu, X. N., Wang, S. Y., Fu, G., Zhang, X. L., Wang, Z. Q., Brindley, P. J., McManus, D. P., Xue, C. L., Feng, Z., Chen, Z., and Han, Z. G. (2003) Nat. Genet. 35, 139–147[CrossRef][Medline] [Order article via Infotrieve]
  36. Cohen, L. S., Mikhli, C., Friedman, C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E., and Davis, R. E. (2004) RNA (N. Y.) 10, 1609–1624
  37. Lewis, F. A., Stirewalt, M. A., Souza, C. P., and Gazzinelli, G. (1986) J. Parasitol. 72, 813–829[CrossRef][Medline] [Order article via Infotrieve]
  38. Lewis, F. A. (1998) in Current Protocols in Immunology (Coligan, J. E., Kruisbeek, J. A., Margulies, D. E., Shevach, E. M., and Strober, W., eds) pp. 19.1.1–19.1.28, John Wiley & Sons, Inc., New York
  39. Kawanaka, M., Hayashi, S., and Ohtomo, H. (1983) J. Parasitol. 69, 991–992[CrossRef][Medline] [Order article via Infotrieve]
  40. Bixler, L. M., Lerner, J. P., Ivanchenko, M., McCormick, R. S., Barnes, D. W., and Bayne, C. J. (2001) J. Parasitol. 87, 1167–1168[CrossRef][Medline] [Order article via Infotrieve]
  41. Davis, R. E., Parra, A., LoVerde, P. T., Ribeiro, E., Glorioso, G., and Hodgson, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8687–8692[Abstract/Free Full Text]
  42. Jones, L. J., Yue, S. T., Cheung, C. Y., and Singer, V. L. (1998) Anal. Biochem. 265, 368–374[CrossRef][Medline] [Order article via Infotrieve]
  43. Mankad, R. V., Gimelbrant, A. A., and McClintock, T. S. (1998) Biol. Bull. 195, 251–254[Abstract]
  44. Kozak, M. (1991) J. Cell Biol. 115, 887–903[Abstract/Free Full Text]
  45. Cavener, D. G., and Ray, S. C. (1991) Nucleic Acids Res. 19, 3185–3192[Abstract/Free Full Text]
  46. Peri, S., and Pandey, A. (2001) Trends Genet. 17, 685–687[CrossRef][Medline] [Order article via Infotrieve]
  47. Pesole, G., Bernardi, G., and Saccone, C. (1999) FEBS Lett. 464, 60–62[CrossRef][Medline] [Order article via Infotrieve]
  48. Wang, X. Q., and Rothnagel, J. A. (2004) Nucleic Acids Res. 32, 1382–1391[Abstract/Free Full Text]
  49. Kozak, M. (1999) Gene (Amst.) 234, 187–208[CrossRef][Medline] [Order article via Infotrieve]
  50. Kozak, M. (2002) Gene (Amst.) 299, 1–34[CrossRef][Medline] [Order article via Infotrieve]
  51. Wang, X. C., Yang, J., Huang, W., He, L., Yu, J. T., Lin, Q. S., Li, W., and Zhou, H. M. (2002) Int. J. Biochem. Cell Biol. 34, 983–991[CrossRef][Medline] [Order article via Infotrieve]
  52. Enjolras, N., Rodriguez, M. H., Plantier, J. L., Maurice, M., Attali, O., and Negrier, C. (1999) Thromb. Haemostasis 82, 1264–1269[Medline] [Order article via Infotrieve]
  53. Fernandez, C., Gregory, W. F., Loke, P., and Maizels, R. M. (2002) Mol. Biochem. Parasitol. 122, 171–180[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/2/733    most recent
M506963200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, G.
Right arrow Articles by Davis, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, G.
Right arrow Articles by Davis, R. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement