The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

Abstract

Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes.

INTRODUCTION

Eukaryotic cells use three different RNA polymerases (designated Pol I, Pol II, and Pol III) to transcribe their nuclear genome. Each of the three RNA polymerases transcribes specific classes of genes. Pol I synthesizes the large rRNA precursor, while Pol II produces mRNAs, most snRNAs, and snoRNAs (1, 2). Pol III synthesizes tRNAs, 5S rRNA, U6 snRNA, and several other small RNA molecules (3).

Leishmania and other trypanosomatids, such as Trypanosoma cruzi and Trypanosoma brucei, possess the three typical RNA polymerases (4, 5). However, these protozoan parasites present unusual mechanisms of gene expression, including Pol II polycistronic transcription (6, 7). The genomes of these organisms are organized into large polycistronic gene clusters (PGCs), i.e., 10s to 100s of protein-coding genes arranged sequentially on the same strand of DNA. Most chromosomes contain at least two PGCs, which can be either divergently transcribed (toward the telomeres) or convergently transcribed (away from the telomeres). Pol II transcription of an entire PGC initiates at a single region located upstream of the first gene of the cluster (8–10), and mature nuclear mRNAs are generated from primary transcripts by trans splicing and polyadenylation (11). trans Splicing is a process that adjoins a capped 39-nucleotide miniexon or spliced leader (SL) to the 5′ termini of all the mRNAs (12, 13). The most conserved sequences needed for this process are an AG dinucleotide at the 3′ splice site and an upstream pyrimidine-rich region (14–17). The trans splicing and polyadenylation of contiguous genes are linked, as the selection of a splice site for a gene influences the choice of a polyadenylation site for the upstream gene (18).

Pol III transcription in trypanosomatids is also atypical, as this enzyme transcribes all snRNA genes (not only U6 snRNA) in these organisms (19). In Leishmania major, most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes, and the majority of the clusters are located at the boundaries of PGCs (20, 21). However, some tRNA genes are single genes that are not part of a cluster, and some of them are located inside PGCs. One such gene is the selenocysteine (Sec) tRNA (tRNA-Sec) gene, which is embedded into a large cluster of protein-coding genes on chromosome 6.

Selenocysteine, the 21st amino acid, is present in a group of proteins known as selenoproteins in bacteria, archaea, and eukarya (22). The presence of selenoproteins and all the machinery required for its synthesis has been demonstrated in L. major and other trypanosomatids (23, 24). Sec insertion is directed by a UGA codon, which is usually a stop codon, assisted by a specific structural signal located in the 3′ untranslated region of the mRNA (25). A special tRNA species, tRNA-Sec, inserts Sec into nascent selenoproteins. Like other eukaryotic tRNA genes, tRNA-Sec is transcribed by Pol III in vertebrates. One of the typical characteristics of most tRNA genes is that their promoter sequences are internal and consist of two conserved elements: boxes A and B (26). Nevertheless, in Xenopus laevis and other vertebrates, transcription of tRNA-Sec genes is directed by an internal box B and three extragenic domains: a TATA box, a proximal sequence element, and an activator element (27, 28).

The consensus sequences of trypanosomatid tRNA promoter elements were determined by analyzing the sequences of all tRNA genes in L. major, T. brucei, and T. cruzi and comparing them to the sequences of boxes A and B from Saccharomyces cerevisiae (21, 29). Analysis of the promoter sequences from tRNA-Sec genes in trypanosomatids indicated that box A contains an additional A residue between bases 2 and 3 (TGAGCTCAGCTGG, in which the additional A residue is underlined) compared with the consensus sequence (TGGCTCAGCTGG) (21). A similar insertion was previously reported in tRNA-Sec genes from other organisms (30). Regarding box B, tRNA-Sec genes from trypanosomatids present two changes (CGTTCGATTCG, in which the two changes are underlined) compared to the highly conserved consensus sequence (GGTTCGANTCC): a C (instead of a G) at position 1 and a G (in place of a C) at position 11. In other species, the sequence of box B from tRNA-Sec is identical to the corresponding consensus sequence. Since the sequences of both internal control elements from tRNA-Sec genes in trypanosomatids differ from the corresponding consensus sequences, it was hypothesized that the synthesis of tRNA-Sec is regulated by external elements in these organisms (21). In fact, it was demonstrated that the tRNA-Sec gene in T. brucei is transcribed by Pol II (31).

In the work described here, we performed a transcriptional analysis of the tRNA-Sec gene in L. major. Our data show that, similar to the findings for T. brucei, the L. major tRNA-Sec gene is polycistronically transcribed by Pol II, generating transcripts that contain the miniexon and a poly(A) tail. The same result was observed in T. cruzi, indicating that the participation of Pol II in the transcription of the tRNA-Sec gene might represent a hallmark of trypanosomatids. Interestingly, nuclear run-on data show that Pol III also transcribes the tRNA-Sec gene in L. major. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo and reveal that the relationship between Pol II and Pol III is more complex than was previously believed.

MATERIALS AND METHODS

In silico analysis.

Information for sequence analysis and synteny maps of species of Leishmania (L. major, L. donovani, L. infantum, L. braziliensis, L. mexicana, and L. tarentolae) and Trypanosoma (T. cruzi, T. brucei, T. vivax and T. congolense) was obtained from the TriTrypDB databases (version 8.1; http://tritrypdb.org/tritrypdb/). Sequence comparisons were performed using the ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Culture of L. major and T. cruzi.

Promastigotes from L. major MHOM/IL/81/Friedlin (LSB-132.1) were grown in BM medium (1× M199 medium, pH 7.2, containing 10% heat-inactivated fetal bovine serum, 0.25× brain heart infusion, 40 mM HEPES, 0.01 mg/ml hemin, 0.0002% biotin, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1× l-glutamine) at 26°C and harvested in the mid-log phase. Epimastigotes of T. cruzi CL Brener were maintained in liver infusion-tryptose (LIT) medium containing 10% heat-inactivated fetal bovine serum, 50 IU/ml penicillin, 50 μg/ml streptomycin, and 0.025 mg/ml hemin at 28°C, as previously described (32).

5′-RACE analysis.

5′ rapid amplification of cDNA ends (5′-RACE) experiments were performed with 5 μg of total RNA from L. major or T. cruzi with a kit from Life Technologies, Inc. For the L. major tRNA-Sec gene, the first-strand cDNA was synthesized with primer tRNA-SECgsp1 (5′-TGGCACGCCACGAAG) and the PCR amplifications were performed with the nested primer tRNA-SECgsp2 (5′-ATCGAACGGCTGTGAGAGCA) and the nested abridged anchor primer (AAP; 5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG). For the L. major tRNA-Asp gene (LmjF.24.TRNAASP.01), the first-strand cDNA was synthesized with primer Lm24Asp-GSP1 (5′-CCGGCCGGGAATTGAAC) and the PCR amplifications were carried out with primer Lm24Asp-GSP2 (5′-GGGTCACCCGCGTGACAGGC) and the nested AAP. For the tRNA-Pro gene (LmjF.24.TRNAPRO.01) from L. major, the cDNA was produced with primer Lmc24-ProGSP1 (5′-GGGCCGCTAGGGGAATTGAA) and the PCR amplifications were performed with primer Lmc24-ProGSP2 (5′-TGACCTCCCGCACCCGAAG) and AAP. For the T. cruzi tRNA-Sec gene, the first-strand cDNA was synthesized with primer Nested(dT) (5′-CCTCTGAAGGTTCACGGATCCACATCTAGATTTTTTTTTTTTTTTTTTVN) and two PCR amplifications were performed. The first PCR was performed with primers ME23 (5′-CGCTATTATTGATACAGTTTCTG) and Tb-tRNASec-GSP1 (5′-CACCACAAAGGCCGA). The second PCR amplification was performed using the first PCR product as the template and primers Tc-tRNASec-GSP2 (5′-AACGGCTGCGAGTCCAAC) and ME23. The nested PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced.

RT-PCR assays.

To map polyadenylation sites for the tRNA-Sec genes from L. major and T. cruzi and for the LmjF.06.0210 gene from L. major, reverse transcription-PCR (RT-PCR) experiments were performed using cDNA prepared with oligonucleotide Nested(dT). For the L. major and T. cruzi tRNA-Sec genes, the first PCR was carried out with primers Lm-Sec1 (5′-AGCCGCGATGAGCTCAGCT) and B1 (5′-CCTCTGAAGGTTCACGGAT) and the second PCR was done with primers Lm-Sec2 (5′-TGGGTGCGGGCTTCAAA) and B2 (5′-CACGGATCCACATCTAGAT). For the LmjF.06.0210 gene, the first PCR was performed with primers Lmj06.0210PA1 (5′-AGCCGACTCATACTGCGGCT) and B1 and the second PCR was done with primers Lmj06.0210PA2 (5′-CTCATGCACTTTAAGCTGTA) and B2. The miniexon addition site for the LmjF.06.0200 gene from L. major was also located by RT-PCR. The cDNA was prepared with oligonucleotide Lmj06.0200.ME1 (5′-AGAGCGACACCCGTGACTTC), and PCR was performed with primers Lmj06.0200ME2 (5′-ACGGAACCCAGAACGCAGGA) and miniexon (5′-AACGCTATATAAGTATCAGTT). The final PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced. Pol III transcription termination sites were mapped by poly(A) tailing of total RNA. For this purpose, 2 μg of total RNA was mixed with 1 μl of 25 mM ATP, 2 μl of 5× poly(A) polymerase reaction buffer (USB), and 1,200 units of S. cerevisiae yeast poly(A) polymerase (USB) in a final volume of 20 μl. The mixture was incubated for 20 min at 37°C, and the reaction was terminated by heating at 65°C for 10 min. The cDNA was prepared with oligonucleotide Nested(dT). The first PCR was done with primers LmSec1 (5′-AGCCGCGATGAGCTCAGCT) and B1, and the second PCR was done with primers LmSec2 (5′-TGGGTGCGGGCTTCAAA) and B2.

Northern blot analysis.

Total RNA was isolated using the TRI Reagent (Sigma), as specified by the supplier. RNA (12 μg) was separated on 10% polyacrylamide–8% urea gels. After electrophoresis, nucleic acids were transferred to Hybond N+ membranes (Amersham) by electroblotting using a Trans-Blot semidry system. The tRNA-Sec probe corresponds to a 190-bp fragment that includes the 88 bp of the tRNA-Sec gene plus 86 bp of the 5′ upstream region, and that was labeled with [α-³²P]dCTP using a High Prime labeling system (Amersham). For tRNA-Lys, primer TRNA-Lysc03-GSP1 (5′-GCGCACTCCGTGGGG) was labeled with [γ-³²P]ATP by T4 kinase. Hybridizations were performed in 6× SSPE (60 mM Na₂HPO₄, 0.9 M NaCl, 6 mM EDTA), 5× Denhardt's reagent, and 1% SDS at 42°C. Washing was carried out at 55°C in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS.

Molecular cloning into pGEM-T Easy.

DNA fragments from L. major chromosome 6 were amplified by PCR and cloned into the pGEM-T Easy vector. LmjF.06.0370 (521 bp) was amplified with oligonucleotides Lm06-0370-5′ (5′-GAAGCGATGGACTGTTCTGG) and Lm06-0370-3′ (5′-CGGTCCTTGCTGCGAATATC), and LmjF.06.0360 (539 bp) was amplified with primers Lm06-0360-5′ (5′-CTCCTCTTCTGGACATTTGCT) and Lm06-0360-3′ (5′-TTCCCTCCACTTGCAACATAG). LmjF.06.0350 (500 bp) was amplified with oligonucleotides Lm06-0350-5′ (5′-CGGTTCAACGGTAGTCTCTTC) and Lm06-0350-3′ (5′-TAGAGGAACCAGAACGGGTAG), and LmjF.06.0340 (503 bp) was amplified with oligonucleotides Lm06-0340-5′ (5′-CCTCGCATACACCCTTTCGG) and Lm06-0340-3′ (5′-CGCGAATGTACACCACACGG). LmjF.06.0260 (500 bp) was amplified with oligonucleotides Lm06-0260-5′ (5′-CCTGCTTGCTGTCGATGGTG) and Lm06-0260-3′ (5′-TCGCCTCATCCTCCTCTTGC), and LmjF.06.0210 (503 bp) was amplified with oligonucleotides Lm06-0210-5′ (5′-GCCGGAGACATTTGCGTAC) and Lm06-0210-3′ (5′-CTATGGCGACGGGATCATC). LmjF.06.0200 (547 bp) was amplified with oloigonucleotides Lm06-0200-5′ (5′-CCATCCCATGACAAGAGC) and Lm06-0200-3′ (5′-TGTAGTCGCTGTACTCGC), and LmjF.06.0110 (554 bp) was amplified with oligonucleotides Lm06-0110-5′ (5′-TTCACTACCGCGATAGGGTTG) and Lm06-0110-3′ (5′-CCATATCCAGATCCTGCATCC). The tRNA-Sec gene (524 bp) was amplified with oligonucleotides Lm-TRNASEC524-5′ (5′-CCGGCTGCCTTCATCAACTC) and Lm-TRNASEC524-3′ (5′-GCGCATACGTTTCGGAGTCC), and the tRNA-Asp gene (369 bp) was amplified with oligonucleotides Lm24-TRNAASP-5′ (5′-GAATGCGCTGCTGAGTCTCT) and Lm24-TRNAASP-3′ (5′-GCGGTATGCGTGTTGGTGTA). The tRNA-Phe gene (LmjF.09.TRNAPHE.01; 338 bp) was amplified with oligonucleotides Lm09-TRNAPHE-5′ (5′-TTCATCCGCGCAAAGAGG) and Lm09-TRNAPHE-3′ (5′-GGCCTTCCACGTATTTCG), and the tRNA-Pro gene (352 bp) was amplified with oligonucleotides Lm24-TRNAPRO-5′ (5′-GCGATCTCGTGGCTCTGGAG) and Lm24-TRNAPRO-3′ (5′-ACAGCTCATCCAACGGGCGC). The tRNA-Tyr gene (LmjF.36.TRNATYR.01; 316 bp) was amplified with oligonucleotides Lm36-TRNATYR-5′ (5′-AGTGCCGAGAAGTTCGACG) and Lm36-TRNATYR-3′ (5′-TCGTCTCCGTTCCTGTTGC), and the 5S rRNA gene (LmjF.15.5SrRNA.01; 344 bp) was amplified with oligonucleotides Lm15-rRNA5S-5′ (5′-GAAAGCATCTCTGTGGGTTCGA) and Lm15-rRNA5S-3′ (5′-CCCGGGGTCCTGCAAATG). The 18S rRNA gene (LmjF.27.rRNA.01; 370 bp) was amplified with oligonucleotides Lm-rRNA18S-5′ (5′-CGGCCTCTAGGAATGAAGG) and Lm-rRNA18S-3′ (5′-CCCCTGAGACTGTAACCTC). The α-tubulin gene (338 bp) was amplified with primers alfa-tub-5′ (5′-AGAAGTCCAAGCTCGGCTACAC) and alfa-tub-3′ (5′-GTAGTTGATGCCGCACTTGAAG). After transformation of JM109 competent cells, plasmid DNA was purified from colorless colonies with NucleoSpin plasmid columns (Macherey-Nagel) as specified by the supplier. The identity of each insert was confirmed by sequencing using the T7 and SP6 primers.

Molecular cloning into M13 and preparation of single-stranded DNA.

DNA fragments from tRNA-Sec, tRNA-Tyr, tRNA-Pro, 5S rRNA, LmjF.06.0200, LmjF.06.0210, 18S rRNA, and α-tubulin were excised from their respective constructs in pGEM-T Easy and cloned into M13mp18 and M13mp19 replicative-form DNA. The tRNA-Tyr, tRNA-Pro, 5S rRNA, LmjF.06.0200, LmjF.06.0210, and 18S rRNA sequences were cloned into M13 SacI and SphI sites. The tRNA-Sec and α-tubulin sequences were cloned into M13 SalI and SphI sites. After transformation of Escherichia coli JM109 cells (Promega), single-stranded DNA was purified from colorless plaques with QIAprep Spin M13 columns (Qiagen) as specified by the supplier.

Nuclear run-on assays.

Nuclei were isolated from 2.5 × 10⁸ L. major promastigotes by washing twice in phosphate-buffered saline (PBS), resuspending the cells in 4 ml ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 3 mM CaCl₂, 2 mM MgCl₂), and adding NP-40 to a final concentration of 0.5%. Cells were transferred to a Dounce homogenizer and broken with 40 strokes; the nuclei were collected by centrifugation (1,400 × g) and washed once with lysis buffer. RNA elongation was performed as described elsewhere (8, 33). Briefly, nuclei were resuspended in 100 μl of run-on mix: 100 mM Tris-HCl (pH 7.5), 25% glycerol, 0.15 mM spermine, 0.5 mM spermidine, 2 mM dithiothreitol, 40 U RNasin (Promega), 2 mM MgCl₂, 4 mM MnCl₂, 50 mM NaCl, 50 mM KCl, 2 mM ATP, 2 mM GTP, 2 mM UTP, 10 μM CTP, and 250 μCi of [α-³²P]CTP (3,000 Ci/mmol; Amersham). The incubation was carried out for 6 min at 26°C, after which DNase I (10 U) was added. Incubation was continued for 5 min at 37°C and then stopped by the addition of 100 μl of 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS, and 100 μg/ml proteinase K. After 15 min incubation at 37°C, RNA was extracted with phenol-chloroform and separated from free nucleotides by G-50 Sephadex chromatography. Labeled nascent RNA was hybridized to Hybond filters (Amersham) containing dots of 2 μg of plasmid DNA (for the experiments whose results are shown in Fig. 3, 4, and 6) or single-stranded M13 DNA (for the experiments whose results are shown in Fig. 5). Hybridization was performed for 48 h at 50°C in 50% formamide, 5× SSC, 0.2% SDS, 4× Denhardt's reagent, and 100 μg/ml salmon sperm DNA. Posthybridization washes were carried out in 0.1× SSC and 0.1% SDS at 65°C. In the assays carried out in the presence of UV light, promastigotes (in a total volume of 15 ml) were irradiated in petri dishes, with agitation, in a Stratalinker UV cross-linker (Stratagene). After irradiation, cells were incubated for at least 1.5 h at 28°C to allow the clearing of RNA polymerases engaged prior to irradiation. Elongation of nascent RNA in the presence of transcription inhibitors was performed by preincubating the nuclei with α-amanitin (Roche Molecular Biochemicals) or tagetitoxin (Tagetin; Epicentre Biotechnologies) for 15 min on ice in lysis buffer. The nuclei were next pelleted and resuspended in elongation buffer in the presence of the drugs.

FIG 3.

Nuclear run-on analysis of the tRNA-Sec gene. (A) (Top) Labeled nascent RNA from nuclei isolated from L. major promastigotes was hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. (Bottom) A genomic map of the protein-coding genes and the tRNA-Sec gene from L. major chromosome 6 is shown. The locations and sizes of the DNA fragments used in the nuclear run-on assays are indicated by the lines below the map. The Pol II genes analyzed were LmjF.06.0370, LmjF.06.0360, LmjF.06.0350, LmjF.06.0340, LmjF.06.0260, LmjF.06.0210, LmjF.06.0200, and LmjF.06.0110. Also, several genes transcribed by Pol III (tRNA-Asp, tRNA-Phe, tRNA-Pro, tRNA-Tyr, and 5S rRNA), as well as the 18S rRNA gene (transcribed by Pol I), were included. As a control, an empty vector (pG) was also analyzed. (B) Hybridization signals for LmjF.06.0210, LmjF.06.0200, and tRNA-Sec from five independent nuclear run-on experiments. (C) Relative rates of transcription for LmjF.06.0210, LmjF.06.0200, and tRNA-Sec. The dots shown in panel B were quantified and plotted, with the signal obtained with LmjF.06.0210 considered to be 100%. Hybridization signals were divided by the probe length. One-way analysis of variance was performed with the data (F₂ = 13.7; P < 0.001); multiple comparisons by the Holm-Sidak method indicated that the values are significantly different between tRNA-Sec and LmjF.06.0200 (t = 4.46; P = 0.002) and between tRNA-Sec and LmjF.06.0210 (t = 4.60; P = 0.002).

FIG 4.

Effect of UV irradiation on transcription of the tRNA-Sec locus. (A) Results of nuclear run-on assays carried out with nuclei isolated from promastigotes that were irradiated with UV light at three different intensities (1.25, 2.5 and 5 kJ/m², as indicated below each panel). After irradiation, cells were incubated for 1.5 h at 28°C to allow the clearing of RNA polymerases engaged prior to irradiation. The genes analyzed were the same ones indicated in the legend to Fig. 3A. (B) The results shown in panel A and from an independent experiment were quantified, and the transcription signal for each gene relative to that for the nonirradiated control was plotted against UV dose. Values represent the means of two experiments.

FIG 6.

Effect of tagetitoxin on tRNA-Sec gene transcription. (A) Nuclear run-on RNA was radiolabeled in the presence of tagetitoxin at 160 μM and hybridized to dot blots of double-stranded DNAs (2 μg) cloned into the pGEM-T Easy vector. The genes analyzed were tRNA-Sec, LmjF.06.0210, and LmjF.06.0200. Several genes transcribed by Pol III (tRNA-Pro, tRNA-Asp, and 5S rRNA), as well as the 18S rRNA gene (transcribed by Pol I), were also included. As a control, an empty vector (pG) was also analyzed. (B) The results shown in panel A and from an independent experiment were quantified, and the transcription signal for each gene relative to that for the control was plotted. Values represent the means from two experiments. All RNA levels were normalized to the level of 18S rRNA.

FIG 5.

α-Amanitin sensitivity of tRNA-Sec gene transcription. (A) Nuclear run-on RNA was radiolabeled in the presence of different doses of α-amanitin (0, 100, 200, and 400 μg/ml) and hybridized to filters containing single-stranded DNAs of tRNA-Sec, LmjF.06.0210, and LmjF.06.0200. The genes used as controls were tRNA-Tyr, tRNA-Pro, 5S rRNA (Pol III), α-tubulin (TUB; Pol II), 18S rRNA (Pol I), and the mp18 vector with no insert (M13). Lanes S, DNA complementary to the sense strand; lanes A, DNA complementary to the antisense strand. (B) Signals obtained with DNA complementary to the sense strand in panel A and from an independent experiment were quantified relative to the signal for the control (α-amanitin dose, 0 mg/ml) and plotted against the α-amanitin dose. Values represent the means from two experiments. All RNA levels were normalized to the level of 18S rRNA.

RESULTS

Genomic location of the tRNA-Sec gene in L. major and other trypanosomatids.

The majority of tRNA genes in L. major are clustered with other tRNA genes or other genes transcribed by Pol III (20, 21). These clusters are frequently located at the boundaries of PGCs, which are transcribed by Pol II. However, the tRNA-Sec gene is a single gene that is inserted into the largest PGC of chromosome 6 (Fig. 1A). In order to examine the genomic context of the tRNA-Sec genes in other species of Leishmania, we analyzed the genomic databases of several species recently sequenced: L. infantum (strain JPCM5), L. braziliensis (M2904) (34, 35), L. mexicana (U1103) (35), L. donovani (BPK282/0cl4) (36), and L. tarentolae (Parrot-TarII) (37). We found that in all the Leishmania species the tRNA-Sec gene is a single-copy gene embedded into the same PGC of chromosome 6 (data not shown). Thus, synteny of the tRNA-Sec locus is observed among Leishmania species.

FIG 1.

Synteny of the tRNA-Sec loci in trypanosomatids. The genomic context of the tRNA-Sec genes is shown for L. major (Lmaj) (A) and for T. cruzi (Tcru), T. brucei (Tbru), T. vivax (Tviv), and T. congolense (Tcon) (B). Orthologous genes are joined by gray lines. Enlargements of the tRNA-Sec gene in L. major and one of the tRNA-Sec genes in T. cruzi are shown, indicating the miniexon addition site (AG) and the poly(A) region (PA) of the tRNA-Sec genes. In L. major, the positions of the poly(A) region for LmjF.06.0210 and AG for LmjF.06.0200 are indicated. The positions of the primers used to map processing signals are also denoted with arrows. In panel B, genes A correspond to TcCLB.506467.50 and TcCLB.506467.40 (T. cruzi), Tb927.9.2350 (T. brucei), TvY486_0900740 (T. vivax), and TcIL3000_0_10490 (T. congolense). Genes B correspond to TcCLB.506467.29, Tb927.9.2320, and TvY486_0900730. Genes C correspond to Tb927.9.2390 and TcIL3000_0_10500. Gene D corresponds to TvY486_0900750, and gene E corresponds to TcIL3000_0_10480. b, number of bases.

In T. brucei there are two copies of the tRNA-Sec gene located on chromosome 9 (21, 38). Analyses of the genomic databases of T. cruzi (CL Brener) (39), T. vivax (Y486), and T. congolense (IL-3000) (40) showed that the presence of two tRNA-Sec genes is conserved across the genus Trypanosoma (Fig. 1B). Although the tRNA-Sec locus is syntenic in these species, several differences were observed; for instance, the Ser/Thr protein kinase gene (gene A in Fig. 1B) is duplicated in T. cruzi.

tRNA-Sec transcripts contain the miniexon and a poly(A) tail in L. major and T. cruzi.

The presence of atypical boxes A and B in the tRNA-Sec gene in L. major and its location inside a cluster of protein-coding genes suggest that the mechanisms that regulate the transcription of this gene might be different from those that regulate the transcription of other tRNA-Sec genes. To explore this possibility, we performed 5′-RACE experiments in order to map the transcription start sites of the tRNA-Sec gene. From this assay, we obtained two DNA bands of approximately 300 and 225 bp and a smaller and fainter band of about 150 bp contained within a smear (Fig. 2A). These bands were cloned and sequenced. Interestingly, analysis of several clones from the largest DNA band showed the presence of the miniexon sequence at the 5′ end, located 150 bp upstream of the tRNA-Sec gene (Fig. 2B), which suggested that the tRNA-Sec gene might be transcribed by Pol II, as if it were a protein-coding gene. Indeed, it was reported that the tRNA-Sec gene in the related parasite T. brucei is transcribed by Pol II (31).

FIG 2.

Mapping of processing sites and Northern blot analysis of the tRNA-Sec gene in L. major. (A) The final products of a 5′-RACE experiment of the tRNA-Sec gene were analyzed on a 1.5% agarose gel (lane 1). The size marker corresponds to a 1-kb ladder (Invitrogen) (lane M). The sequences of the tRNA-Sec gene and flanking regions for L. major (B) and T. cruzi (E) are shown. In both cases, the tRNA-Sec gene is highlighted in gray. Upstream of the genes, the miniexon acceptor sites (AG) and the position of all clones found in the 5′-RACE analyses are shown in bold type and marked with an arrow with a number that indicates the total number of clones with the indicated sequences found at that position. Pyrimidine-rich regions found upstream of the miniexon acceptor sites are underlined. Polyadenylation sites found downstream of the tRNA-Sec genes are shown in bold type and marked with an arrow with a number that indicates the total number of clones with the indicated sequences found at that position. The clusters of T residues located downstream from the tRNA-Sec genes are shown in bold type. Internal boxes A and B are labeled and shown in bold type and underlined. For L. major, upstream boxes B and A-like are also labeled and shown in bold type and underlined. (C and D) Hybridization of RNA with a tRNA-Sec-specific probe (C) and a tRNA-Lys-specific probe (D).

Sequence analysis of clones from the 225-bp band showed non-trans-spliced transcripts whose sequences extended 68 or 86 bases upstream of the tRNA-Sec gene. These clones might represent intermediates between the miniexon-containing transcripts and the mature tRNA-Sec. Finally, sequence analysis of clones obtained from the 150-bp band revealed RNA molecules with a 5′ end that corresponded to the mature tRNA-Sec, as well as some smaller molecules. Interestingly, the sequences of two of the clones extended 5 and 9 bp upstream of the mature tRNA's 5′ end (see below) (Fig. 2B).

Since the tRNA-Sec transcripts were found to bear the miniexon sequence, we wondered if they also possessed a poly(A) tail. To determine if the tRNA-Sec transcripts in L. major are polyadenylated, an RT-PCR assay was performed. Only one band was identified and cloned. Analysis of the cloned sequences demonstrated that some tRNA-Sec molecules indeed contain a poly(A) tail, which was added at different positions between 4 and 67 bases downstream of the tRNA-Sec gene (Fig. 2B). Thus, our results show that some of the tRNA-Sec transcripts in L. major contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end. Consequently, the data suggest that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation.

If the tRNA-Sec is indeed polycistronically transcribed, it should be possible to demonstrate the presence of precursor transcripts. To examine this possibility, total RNA was hybridized with a tRNA-Sec probe. As shown in Fig. 2C, RNAs of approximately 80, 400, 650, and 1,000 bases were observed. The ∼80-base band represents the mature tRNA-Sec gene, while the ∼400-base RNA most likely corresponds to the transcript that contains the miniexon and a poly(A) tail of about 130 nucleotides. The larger bands represent longer RNA precursors. To demonstrate that the presence of large precursors was not a general feature of tRNA transcripts in Leishmania, a similar experiment was performed to detect transcripts using a tRNA-Lys-specific probe (transcribed by Pol III), finding only the mature tRNA and not any larger bands (Fig. 2D).

A typical AG sequence was identified as the miniexon attachment site in the transcript of the L. major tRNA-Sec gene (Fig. 2B). To gain insight into other processing signals in the polycistronic transcript, RT-PCR experiments were performed to localize the polyadenylation site of the gene (LmjF.06.0210) located upstream of the tRNA-Sec gene and the miniexon addition site of the downstream gene (LmjF.06.0200). The poly(A) tail of LmjF.06.0210 was found 383 bases downstream of its stop codon, and the miniexon addition site of LmjF.06.0200 was located 203 bases upstream of the start codon of the gene (Fig. 1A). Both sites correspond to processing sites reported on the TriTrypDB web page. However, the LmjF.06.0210 poly(A) site that we found is not reported to be the dominant one, which was located 1,942 bases downstream of the stop codon. This clone might actually represent a dicistronic precursor that contains both LmjF.06.0210 and the tRNA-Sec gene, taking into consideration the fact that the tRNA-Sec gene is located 920 bases downstream of the LmjF.06.0210 stop codon. Pyrimidine-rich regions, which are required for both trans splicing and polyadenylation, were located between the polyadenylation region of LmjF.06.0210 and the miniexon addition site of the tRNA-Sec gene and between the polyadenylation region of the tRNA-Sec gene and the miniexon addition site of LmjF.06.0200. Therefore, canonical signals seem to regulate the processing of the polycistronic transcript of the tRNA-Sec gene in L. major.

Our results showed the participation of Pol II in the synthesis of the tRNA-Sec gene in L. major, similar to what has been reported in T. brucei (31). Therefore, it is likely that this might represent a more generalized feature of gene expression in trypanosomatid species. We thus determined if the tRNA-Sec gene of T. cruzi is also trans spliced and polyadenylated. As predicted, sequence analysis of several clones obtained by 5′-RACE and RT-PCR assays demonstrated the presence of the miniexon 95 bases upstream of the tRNA-Sec gene and the presence of a poly(A) tail between 36 and 39 bases downstream of the tRNA-Sec gene (Fig. 1B and 2E). Thus, the participation of Pol II in transcription of the tRNA-Sec genes seems to be a hallmark of trypanosomatids.

The results of the experiments described above demonstrate the participation of Pol II in transcription of the tRNA-Sec gene in L. major. However, they do not exclude the possibility that the tRNA-Sec gene can be also transcribed by Pol III, considering that putative Pol III internal promoter elements are present in this gene. As mentioned above, two clones obtained in the 5′-RACE assay contained sequences that extended 5 and 9 bases upstream of the tRNA-Sec; since Pol III transcription of tRNAs usually starts a few nucleotides upstream of the tRNA gene, it is possible that these clones represent Pol III transcription start sites. Moreover, sequence analysis of the downstream region of tRNA-Sec showed the presence of two runs of 4 T residues separated by a CC sequence (Fig. 2B), a typical sequence that is associated with the termination of transcription by Pol III. Thus, to determine the presence of clones that end within the runs of T residues located downstream of the tRNA-Sec gene, RT-PCR was performed with total RNA that was poly(A) tailed in vitro. Interestingly, some clones were found to end within the first run of T residues (data not shown), suggesting the participation of Pol III in transcription of the tRNA-Sec gene in L. major. Transcription of tRNA-Sec genes in vertebrates is regulated by three sequence elements located upstream of the gene: a TATA box at about position −30, a proximal sequence element located at about position −70, and an activator element located at position −200 (28). These promoter elements are not contained in the upstream region of the tRNA-Sec gene in L. major. However, we found a consensus box B (GGTTCGATTCC) located 34 bases upstream of the tRNA-Sec gene and a box A-like sequence (TGGCTGCAACGG) located 74 bases upstream of the gene (Fig. 2B). These sequence elements are not present in the tRNA-Sec genes from T. brucei and T. cruzi (Fig. 2E and data not shown).

Nuclear run-on analysis of the tRNA-Sec locus.

In order to analyze nascent transcripts from the tRNA-Sec gene and several protein-coding genes from the same polycistronic unit on chromosome 6, a nuclear run-on assay was carried out (Fig. 3A). The Pol II genes analyzed were LmjF.06.0360, LmjF.06.0350, LmjF.06.0340, LmjF.06.0260, LmjF.06.0210, LmjF.06.0200, and LmjF.06.0110. The LmjF.06.0370 gene, from the adjacent PGC, was also analyzed. As controls, several Pol III genes (tRNA-Asp, tRNA-Phe, tRNA-Pro, tRNA-Tyr, and 5S rRNA), as well as the 18S rRNA gene, which is transcribed by Pol I, were included.

As shown in Fig. 3A, the intensity of the signal obtained with the tRNA-Sec gene was stronger than that obtained with the protein-coding genes that form part of the same polycistronic unit. Moreover, the tRNA-Sec signal was very similar to that observed with other tRNAs and 5S rRNA. The experiment was repeated five more times, and the results obtained each time were very similar. On average, the signal of the tRNA-Sec gene was ∼3.1 times higher than that of the genes that are located directly upstream and downstream of the tRNA-Sec gene and that are part of the same polycistronic unit (Fig. 3B and C). Although the lengths of the three probes analyzed in the experiment whose results are presented in Fig. 3B and C were very similar (524, 547, and 503 bp for tRNA-Sec, LmjF.06.0200, and LmjF.06.0210, respectively), the hybridization signals were divided by the probe length. The G+C contents of the three fragments were also very similar: 60.6, 68.1, and 63.2% for tRNA-Sec, LmjF.06.0200, and LmjF.06.0210, respectively. Thus, these results suggest that tRNA-Sec has another source of transcription, most likely Pol III.

As the promoter sequences for Pol III are located within the coding region, with or without some upstream elements, we wanted to assess if the transcription of tRNA-Sec is initiated at or near its locus. Therefore, we irradiated cells with different UV doses in order to arrest transcription elongation by producing pyrimidine dimers in the DNA (41). Irradiation of cells with incremental doses of UV light resulted in a progressive decrease of nascent transcripts, as assessed by nuclear run-on analysis. As expected, the signals of the protein-coding genes and the18S rRNA gene were progressively reduced as the UV dose was increased, while the transcription of Pol III-dependent genes was relatively conserved. Interestingly, the pattern of reduction in the signal intensity of the tRNA-Sec gene was very similar to that observed with other tRNA genes and 5S rRNA and different from the pattern seen with the protein-coding genes from the same polycistronic unit (Fig. 4). For example, at a UV dose of 2.5 kJ/m², the hybridization signals for LmjF.06.0210 and LmjF.06.0200 were reduced by 54% and 66% of the value for the control, respectively, while that for the tRNA-Sec gene was reduced by only 15%, which is similar to the 10 to 34% reduction observed with the Pol III genes. Therefore, this result indicates that part of the transcription of the tRNA-Sec gene originates from a promoter region located very close to (or within) the tRNA-Sec gene.

It has been reported that Pol II and Pol III show different sensitivities to α-amanitin, since low concentrations of the drug inhibit Pol II transcription but have little impact on Pol III transcription (42, 43). Thus, to further examine the participation of Pol II and Pol III in the transcription of the tRNA-Sec gene, nuclear run-on experiments were carried out with different concentrations of α-amanitin. As in trypanosomatids the noncoding strands are transcribed at different levels, these experiments were performed using single-stranded M13 DNA to analyze the transcription of both strands of the tRNA-Sec gene independently. As shown in Fig. 5A and B, the lowest concentration of α-amanitin (100 μg/ml) reduced Pol II-mediated transcription of LmjF.06.0210 and LmjF.06.0200 by 63 and 65% of the value for the control, respectively. In contrast, the hybridization signal for the tRNA-Sec gene was reduced by only 25%. The transcription of tRNA-Pro, 5S rRNA, and tRNA-Tyr was reduced by 27, 28, and 31%, respectively. Overall, the inhibition curve obtained with the tRNA-Sec gene indicates that the transcription of this gene is a little more sensitive to α-amanitin than the transcription of Pol III genes are but not as sensitive as the transcription of LmjF.06.0210 and LmjF.06.0200. Therefore, these results add further evidence to support the participation of Pol III in the transcription of tRNA-Sec in L. major. As expected, the hybridization signal was severely reduced with the highest concentration of α-amanitin (400 μg/ml) for all genes analyzed, with the exception of the 18S rRNA gene. As reported before in L. major (9), the transcription of α-tubulin, which is encoded by 12 genes in this organism, was not as sensitive to α-amanitin as the transcription of single-copy protein-coding genes. Consistent with previous findings (9), strong antisense transcription was detected for Pol III-transcribed genes, the tRNA-Sec gene, and the 18S rRNA gene (Fig. 5A). It is worth noting that the antisense signals for LmjF.06.0210 and LmjF.06.0200 were higher than those observed for most protein-coding genes on chromosomes 1, 3, and 27 (8, 9, 44). However, the signal obtained with the empty vector control was unusually high in these experiments.

To confirm the involvement of Pol III in the transcription of the tRNA-Sec gene, nuclear run-on experiments were performed with tagetitoxin, a Pol III-specific inhibitor (Fig. 6). The hybridization signal for the tRNA-Sec gene was reduced by 70%, while the signals for tRNA-Pro and tRNA-Asp were reduced by 66 and 76%, respectively. A similar reduction was reported for tRNA genes from chromosomes 3 and 27 in L. major (9). 5S rRNA transcription was more sensitive to tagetitoxin, as it was reduced by 85%. Under the conditions used, the transcription of LmjF.06.0210 and LmjF.06.0200 was slightly reduced by 31 and 37%, respectively, while the transcription of the 18S rRNA was not affected. Therefore, these results confirm that Pol III, in addition to Pol II, transcribes the tRNA-Sec gene in L. major.

Are any other tRNA genes transcribed by Pol II in L. major?

In addition to tRNA-Sec, the L. major genome contains two tRNA genes that are independent genes not associated with other Pol III-transcribed genes and that are inserted into clusters of protein-coding genes. The first one is a tRNA-Asp gene (LmjF.24.TRNAASP.01), and the second one is a tRNA-Pro gene (LmjF.24.TRNAPRO.01); these are located on different PGCs of chromosome 24 (21). To explore the possibility that these two tRNA genes are transcribed by Pol II as part of polycistronic clusters, 5′-RACE assays were performed under conditions similar to those used for the tRNA-Sec genes from L. major and T. cruzi. Sequencing of dozens of clones showed that none of them contained the miniexon. The majority of the clones started at the 5′ end of the mature tRNA, but some of them were shorter. A noteworthy finding was that some of them contained sequences that extended 4 to 5 bases upstream of the tRNA gene, which may represent Pol III transcription start sites. The experiment was repeated several times using different conditions, but the results obtained were the same. Therefore, these results suggest that the single tRNA-Asp and tRNA-Pro genes located on chromosome 24 of L. major are not transcribed by Pol II. Consequently, the fact that a single tRNA gene is inserted into a PGC does not seem to be the only requirement for the participation of Pol II in its synthesis. Nevertheless, we cannot rule out the possibility that these two tRNA genes are transcribed by Pol II and that miniexon-containing transcripts are processed very rapidly, so that we were not able to detect them. Alternatively, it is also possible that other unidentified genes transcribed by Pol III are located in the proximities of the tRNA-Asp and tRNA-Pro genes, so that they are not actually single genes but part of clusters of Pol III genes. The intergenic regions that flank these genes are large enough to contain other Pol III genes (3,212 and 654 bp for the tRNA-Pro gene and 390 and 1,642 bp for the tRNA-Asp gene).

DISCUSSION

It has been shown that the transcription of tRNA-Sec genes is different from the transcription of other tRNA genes in several species analyzed. L. major is not the exception, since the data presented here show that the tRNA-Sec gene in this protozoan parasite is transcribed by both Pol II and Pol III. The tRNA-Sec gene is located inside a polycistronic unit on chromosome 6, and Pol II transcribes it as if it were a protein-coding gene. Consequently, some transcripts of the tRNA-Sec gene contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, just like the mRNAs synthesized from the protein-coding genes that flank the tRNA-Sec gene. A characteristic AG dinucleotide was recognized as the miniexon addition site in the tRNA-Sec transcript from L. major (Fig. 2B), and pyrimidine-rich regions were located between the polyadenylation region of LmjF.06.0210 and the miniexon addition site of tRNA-Sec and between the polyadenylation region of tRNA-Sec and the miniexon addition site of LmjF.06.0200, indicating that typical signals regulate the processing of the polycistronic transcript of the L. major tRNA-Sec gene. In T. cruzi, the tRNA-Sec genes are also inserted into a Pol II polycistronic unit, and our data show that their transcripts are processed by trans splicing and polyadenylation, as has been reported for the tRNA-Sec genes in T. brucei (31). Hence, the involvement of Pol II in the transcription of tRNA-Sec genes seems to be a hallmark of trypanosomatids.

Several lines of evidence indicate that Pol III, in addition to Pol II, participates in transcription of the tRNA-Sec gene in L. major: (i) 5′-RACE and RT-PCR analysis showed the presence of transcripts whose sequences extended 5 to 9 bases upstream of the tRNA-Sec gene and transcripts that terminated in the T-residue tract located immediately downstream of the gene; these transcripts correspond to typical tRNA initiation and termination sites, respectively; (ii) in nuclear run-on analysis, the intensity of the signal observed with the tRNA-Sec gene was stronger than that obtained with the protein-coding genes that form part of the same polycistronic unit, and the signal of the tRNA-Sec gene was very similar to that observed with other tRNAs and 5S rRNA; (iii) nuclear run-on experiments with UV-irradiated cells showed that part of the transcription of the tRNA-Sec gene originates from a promoter region located very close to (or within) the tRNA-Sec gene; (iv) the α-amanitin inhibition curve for the tRNA-Sec gene is more similar to the one observed for Pol III genes than to the one observed for Pol II genes; (v) nuclear run-on experiments with tagetitoxin demonstrated that hybridization signals for the tRNA-Sec gene and other tRNA genes were decreased to similar levels (66 to 76%), while transcription of LmjF.06.0210 and LmjF.06.0200 was slightly affected; and (vi) previously reported data obtained by chromatin immunoprecipitation with microarray technology showed the presence of a TATA-binding protein (TBP) enrichment peak in the tRNA-Sec region (45); TBP peaks were present on all Pol III-transcribed genes but not on snoRNA genes, which are polycistronically transcribed by Pol II with the neighboring protein-coding genes (45); although the TBP signal peak found in the region of the tRNA-Sec gene is not as high as the signals found in tRNA gene clusters, it clearly shows the binding of TBP to the tRNA-Sec gene. In T. brucei it has been shown that TBP (TRF4) is part of Pol III transcription factor TFIIIB (as well as a Pol II transcription factor complex that includes SNAPc and TFIIA) (46).

It has been suggested that the signal observed in a nuclear run-on assay partially represents RNA abundance, as well as the transcription rate, reflecting the rapid rate of processing in Leishmania, and that factors such as the size and the G+C content of the fragment and the secondary structure within the RNA may also contribute to the observed differences in hybridization signals between fragments (8). However, the fact that the signals obtained with the tRNA-Sec gene were reproducibly higher than those obtained with other genes from the same polycistronic unit and the observation that the signals obtained with Pol III genes and tRNA-Sec gene are comparable (Fig. 3) strongly indicate that Pol III is also involved in transcription of the tRNA-Sec gene in L. major.

In T. brucei, tRNA-Sec precursors that contain the miniexon and a poly(A) tail require further processing to create the mature tRNA-Sec (31). At present, we do not know whether the tRNA-Sec transcripts that contain the miniexon and a poly(A) tail in L. major are additionally processed to originate mature tRNA-Sec, as in T. brucei, or whether they are degraded. The question of why in T. brucei the tRNA-Sec gene is transcribed only by Pol II while in L. major it is transcribed by both Pol II and Pol III then arises. As T. brucei contains two tRNA genes, it is possible that Pol II transcription of these genes generates the levels of tRNA-Sec that the cell requires, while L. major, which has a single tRNA-Sec gene, might need the additional participation of Pol III to obtain higher levels of tRNA-Sec. Since the tRNA-Sec genes in both species contain identical internal boxes A and B and they contain a T-residue tract at the 3′ end of the genes, it is possible that the upstream box B and box A-like elements, which are not found in T. brucei, help recruit Pol III to the tRNA-Sec gene in L. major.

To our knowledge, this is the first report of a gene transcribed by more than one RNA polymerase in trypanosomatids. Only a few examples have been reported in other organisms. A recent study of human cells by chromatin immunoprecipitation sequencing revealed that the gene encoding the H1 RNA (a component of RNase P), which is considered a type 3 Pol III gene, can be transcribed by either Pol III or Pol II in vivo (47). This gene contains a T-residue tract, which is the typical Pol III termination signal, and a 3′ box, a Pol II termination signal present on snRNAs. Most transcripts that were detected ended at the T-residue tract, suggesting that the RNA transcribed by Pol II is highly unstable. Another example is the human c-myc promoter, which is transcribed by Pol II and Pol III in vitro and in vivo (48). Also, it was recently reported in human that Pol III is able to accurately initiate transcription from Pol II core promoters in in vitro transcription assays (49). The ratio of DNA template to nuclear extract determines whether Pol II, Pol III, or both enzymes start transcription from the Pol II promoter, indicating that polymerase specificity is not constant but instead depends on transcription conditions (49). Another study in S. cerevisiae yeast showed that Pol II transcribes two sequences found in the promoter and the terminator regions of the rRNA genes that overlap the sequences of the 35S rRNA precursor transcribed by Pol I (50). Inhibition of Pol II transcription decreases Pol III transcription of the 5S rRNA gene. Thus, these findings reveal a complex relationship among all three RNA polymerases in the rRNA loci from yeast (50).

Together, our data support the conclusion that the tRNA-Sec gene in L. major is transcribed by Pol II and by Pol III. It would be interesting to measure the ratio of Pol II and Pol III transcripts and to explore if such a ratio changes in different stages of L. major growth or under different growth conditions. Our results suggest that the tRNA-Sec gene in T. cruzi is transcribed by Pol II. It remains to be investigated whether it is also transcribed by Pol III, as in L. major, or if it is transcribed only by Pol II, as in T. brucei. Similar to the situation in other organisms, the relationship between Pol II and Pol III in trypanosomatids seems to be more complex than was originally estimated. Further investigations are required to gain insight into this very important area of gene expression.