The Trypanosomatid Pr77-hallmark contains a downstream core promoter element essential for transcription activity of the Trypanosoma cruzi L1Tc retrotransposon

Abstract

Background: Trypanosomatid genomes are highly colonized by non-LTR retroelements that make up to 5% of the nuclear genome. These elements are mainly accumulated in the strand switch regions (SSRs) where polycistronic transcription is initiated and have a 77 nt-long sequence--Pr77--at their 5' ends. L1Tc is the best represented retrotransposon in the Trypanosoma cruzi genome and is a potentially functional autonomous element that encodes its own retrotransposition machinery. The Pr77 of the T. cruzi L1Tc element activates gene transcription via RNA polymerase II, generating abundant, unspliced transcripts which are translated.

Results: The present manuscript describes the identification of a downstream core promoter element (DPE) in the L1Tc Pr77 sequence. Just four nucleotides long (CGTG), it covers in Pr77 positions +25 to +28 of the described L1Tc transcription start site. The Pr77-DPE motif is conserved in terms of sequence composition and position in the Pr77 of most trypanosomatid non-LTR retrotransposons, independent of the coding or non-coding capacity of these retroelements. Transcription assays in T. cruzi stable transfectants with vector containing point mutations at 17 locations of the Pr77 nucleotide sequence evidence that the DPE motif is essential for the promoter function of Pr77. Furthermore, the obtained data show that other nucleotides also contributed to the promoter function of Pr77. In addition, the presented results indicate that parasite nuclear proteins specifically bind to different regions of the Pr77 sequence although the strongest binding is to the DPE motif. Moreover, it is shown that the DPE sense single-stranded sequence is being required in DNA-protein recognition of nuclear factors.

Conclusions: The Pr77 sequence present in most of non-LTR retrotransposons of trypanosomatids contains a downstream core promoter element (DPE) which is conserved in terms of nucleotide composition and location. The Pr77-DPE motif is essential for the transcriptional activity of Pr77 although other nucleotides are also involved. DPE has a high affinity binding for nuclear proteins in T. cruzi. The wide retroelement-mediated distribution of Pr77 suggests that it may represent an important tool for regulating gene expression in trypanosomatids.

Fig. 1

Diagram of the L1Tc element including the Pr77 sequence. a Diagram of the L1Tc retrotransposon from T. cruzi. The coding sequences for AP endonuclease (AP), reverse transcriptase (RT), RNaseH (RH), the nucleic acid chaperone (NAC) proteins, and a 2A-self cleaving sequence, are shown in boxes. The Pr77 internal promoter is represented by a black flag, and the L1TcRz ribozyme by an empty arrow-head. The sequence of the Pr77 promoter is shown and the DPE motif labelled in bold face. b Alignment of the nucleotide sequences of the 5′ region of the different types of insect retrotransposon and developmentally regulated genes. The transcription start sites are underlined when known. Nucleotides are numbered from the transcription start site position (+1). The DPE motif is indicated in bold. A diagram of the DPE motif consensus (CGTG) with the CGTT variant for the TRAS1 retroelement is shown below the alignment

Fig. 2

Point mutations in the Pr77 sequence in stable transfectants for analysis of the involvement of these nucleotide positions in the transcription capacity of the Pr77 promoter. The DPE motif is shadowed in the Pr77 L1Tc sequence. Nucleotides are numbered from the L1Tc transcription start site (+1). Conserved nucleotides among L1Tc and NARTc from T. cruzi, and ingi and RIME from T. brucei, are indicated by asterisks below the Pr77 sequence. Mutant names (from M1 to M17) are shown on the left hand side and the corresponding nucleotide substitution and position indicated for each mutant. Also on the left hand side it is indicated whether the DNA secondary structure is not affected (NA), slightly affected (+) or very affected (+++) by the point mutation, as predicted by the Mfold program

Fig. 3

Analysis of transcriptional activity of Pr77-derived sequences in T. cruzi stable transfectants by northern blotting. a, b, c, d Cytoplasmic RNA from T. cruzi stable transfectants eletroporated with pTEXPr77Luc or pTEXPr77Mut1-17Luc, was electrophoresed in 1 % denaturing agarose gel, transferred to nylon membranes, and hybridized with ³²P-labelled Luc (luc) and KMP11 (kmp11) coding sequences as probes. The ethidium bromide staining of ribosomal RNAs is shown below each panel (a-d). Cytoplasmic RNA from wild type Y epimastigotes and parasites transfected with the pTEXLuc construct were used as negative and positive controls (C- and C+ respectively). The northern blots were performed three times. The percentage of transcribed Luc mRNA when using the pTEXPr77Luc construct is shown below the luc hybridization panel as TR (%)

Fig. 4

RT-PCR detection of LUC mRNAs in T. cruzi stable transfectants and of the 5′ end nature of Luc mRNAs. Cytoplasmic RNA from T. cruzi stable transfectants eletroporated with pTEXPr77Luc or pTEXPr77Mut1-17Luc were used as templates for reverse transcription and subsequent PCR amplification of the Luc mRNA 5′ end using a primer corresponding to the Pr77 5′ end (Pr77-LUC), or the SL primer (SL-LUC), as sense primers. As a control of RNA quality, the 5′ end of the kmp11 RNA was reverse transcribed and PCR amplified. The pTEXPr77Luc and pGEMTSL-Luc plasmids were used as DNAs template in positive control reactions; no DNA was used in negative control reactions. Mutants are numbered from 1 to 17 (M1-17)

Fig. 5

Determination of the in vivo transcription start site of Luc mRNA by primer extension. Total RNAs from T. cruzi transfected with the pTEXPr77Luc (Pr77), pTEXPr77M5Luc, pTEXPr77M7Luc or pTEXPr77M15Luc constructs (M5, M7 and M15) were used for primer extension of LUC mRNAs employing a γ-ATP³² Luc antisense primer (LUC28rev). The cDNA products were resolved in 8 M urea 8 % polyacrylamide gels. The full length extension products (117 nt in length), consistent with the TSS (FL) of Pr77 (nt +1) and 18-mer radiolabelled oligo LUC28rev, are indicated

Fig. 6

Binding of T. cruzi nuclear proteins to the Pr77, DPE-mutated Pr77, and DPE-bearing sequences. a Double-stranded DNA corresponding to the Pr77 (dsPr77), or Pr77 sequence mutated at the DPE motif (dsPr77M1), were 5′ end radiolabelled with γ-ATP³² and loaded onto native 6 % polyacrylamide gels with (+) or without (−) incubation with T. cruzi nuclear proteins (NP). Competition assays were performed by adding as competitors (C) non-labelled DNAs corresponding to the Pr77 sequence (dsPr77), a mixture of aptamers (dsApt) or dsPr77M1 to the reactions at a ratio of 1:50 or 1:5 as indicated in each panel. b Double-stranded DNA corresponding to the Pr77 sequence bearing the DPE motif (dsDPE, nucleotides 12 to 33 of the Pr77 sequence) was 5′ end radiolabelled with γ-ATP³² and loaded onto native 12 % polyacrylamide gels with incubation (+) or without (−) incubation with T. cruzi nuclear proteins (NP). Competition assays were performed by adding as competitor (C) non-labelled dsDPE DNA to the reactions at a ratio of 1:5. Shifted bands are indicated with an arrowhead, and the free probe indicated as ‘FP’

Fig. 7

Binding analysis of T. cruzi nuclear proteins to DPE-bearing DNA as double strand DNA and single strand DPE-bearing oligos (sense and antisense) in native and heat denatured conditions. a DNA corresponding to the double-stranded DPE-bearing Pr77 sequence (dsDPE, nucleotides 12 to 33 of Pr77), single stranded sense DPE oligo (sDPE), single stranded antisense DPE oligo (asDPE) and the heat denatured sDPE and asDPE oligos, was 5′ end radiolabelled with γ-ATP³² and 80.000 cpm of each radiolabeled probe loaded onto native 12 % polyacrylamide gels with (+) or without (−) incubation with T. cruzi nuclear proteins (NP). The mobility of the highest amount of radiolabelled free probe is indicated as free Probe (FP). Asterisks indicate the shifted bands that are formed when nuclear proteins are incubated with both ³²P- single stranded sense DPE and heat denatured ³²P- single stranded sense DPE. b Binding of nuclear proteins to the Pr77 sequence and competition assays with DPE-bearing DNAs. Double-stranded DNA corresponding to the entire Pr77 sequence (dsPr77 probe) was 5′ end radiolabelled with γ-ATP³² and loaded onto native 6 % polyacrylamide gels with incubation (+) or without (−) incubation with T. cruzi nuclear proteins (NP). Competition reactions were performed by adding to the reaction non-labelled DNAs corresponding to the dsDPE sequence, the single stranded DPE oligo (sDPE) or the single stranded DPE antisense oligo (asDPE) as competitors (C) at ratios of 1:10. Shifted bands are indicated by black arrows on both sides of the gel (►). Gel wells are indicated as ‘w’