Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids

The Telomeric Repeat-containing RNAs (TERRA) participate in the homeostasis of telomeres in higher eukaryotes. Here, we investigated the expression of TERRA in Leishmania spp. and Trypanosoma brucei and found evidences for its expression as a specific RNA class. The trypanosomatid TERRA are heterogeneous in size and partially polyadenylated. The levels of TERRA transcripts appear to be modulated through the life cycle in both trypanosomatids investigated, suggesting that TERRA play a stage-specific role in the life cycle of these early-branching eukaryotes.

TERRA; Trypanosoma brucei telomere; Leishmania telomere; telomere transcription

Authorship

Jeziel D Damasceno

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Ribeirão Preto, SP, BrasilUniversidade de São PauloBrasilRibeirão Preto, SP, BrasilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Ribeirão Preto, SP, Brasil

Gabriel LA Silva

Christian Tschudi

Yale UniversitySchool of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, CT, USAYale UniversityUSANew Haven, CT, USAYale UniversitySchool of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, CT, USA

Luiz RO Tosi ⁺+ Corresponding author: luiztosi@fmrp.usp.br

+ Corresponding author: luiztosi@fmrp.usp.br

AUTHORS’ CONTRIBUTION

JDD - Conceived and performed experiments and wrote the manuscript; GLAS - performed experiments shown in Fig. 1C; CT - conceived experiments and critically revised the manuscript; LROT - conceived experiments, supervised the project and wrote the manuscript.

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Figures

Figures (2)

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Fig. 1
: transcription of UUAGGGn-containing RNAs (TERRA) from Leishmania telomeres. All the experiments in the figure were performed using RNA from promastigote forms. (A) Total RNA (~30 µg) from L. major (Lm; cell lines LT252 and LV39), L. amazonensis (La) and L. braziliensis (Lb) was subjected to Northern blotting and probed with 32P-end labelled (CCCTAA)15 or (TTAGGG)15 oligonucleotides. rRNA ethidium bromide (EtBr) indicates ribosomal RNAs stained with EtBr before transferring to the membrane and serves as loading and quality control of total RNA. (B) Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect TERRA derived from different L. major telomeres; left panel, the diagram indicates the strategy used: the reverse transcription step (RT) was performed with (CCCTAA)15 oligonucleotide (dashed arrow); the cDNA generated was used in a PCR reaction performed with oligonucleotides (arrowheads) specific for the ORF (white arrow) closest to the telomere ((TTAGGG)n); ORFs orientated from the internal portion of the chromosome towards telomeres are indicated as (+); ORFs orientated from the telomeres towards the internal region of the chromosome are indicated as (-); right panel shows EtBr-staining of PCR products after fractionation in agarose gels; the cDNAs used as template for the PCR were generated in reverse transcription reactions performed in the presence (RT+) or absence (RT-) of Reverse Transcriptase; the specific primers for the PCR were designed for ORFs LmjF01.0010, LmjF18.0010, LmjF04.0010, LmjF11.0010, LmjF20.1620 and LmjF23.0030. (C) Left scheme indicates the approach for detection of TERRA by RT-PCR; total RNA (3 µg) from Trypanosoma brucei (Tb; bloodstream form, SM cell line) and L. braziliensis (Lb) was treated with DNAseI and submitted to reverse transcription reaction using oligo(dT) as primer; RT+ and RT- indicates addition or omission of reverse transcriptase enzyme from the cDNA synthesis reaction; the cDNA generated was used as template in a PCR reaction with oligo(dT) and a specific telomere sequence (AGGGTT)4 as primers; right panel shows Ethidium Bromide-staining of PCR products; in the bottom, actin and HTBF genes were used as positive controls for polyadenylated RNA in T. brucei and L. braziliensis, respectively. (D) Total RNA from L. braziliensis was fractionated on an oligo(dT) resin (Oligotex mRNA Mini Kit, QIAGEN); equivalent volumes from Input, flow through (FT), washes and eluted material were analysed by Northern blotting as in (A); samples were probed for a-tubulin as a positive control for the binding of poly(A)+ RNA. (E) Total RNA (~15 µg) from L. braziliensis was left untreated (Ctl) or subjected to polynucleotide kinase treatment (PNK) before incubation with terminator exonuclease (TE); samples were subjected to Northern blot analysis as in (A) and probed for a-tubulin as a control for 5’ CAP-containing RNA; rRNA (MetBlue) refers to rRNA staining with methylene blue after transfer to the membrane and serves as control for 5’mono-phosphorylated RNA. (F) Metacyclic and procyclic parasites were purified as previously described (Sacks et al. 1985). Briefly, stationary-phase L. major (LV39) promastigotes were incubated with the lectin PNA and agglutinated cells (PNA+) were separated from metacyclic parasites (PNA-) by centrifugation; the purification yield was 2.0 to 3.5%; the typical metacyclic morphology was used to confirm the sample purity; total RNA (~50 µg) from PNA+ and PNA- cells were subjected to Northern blot analysis as in (A); TERRA signal was quantified with ImageJ software, normalised with rRNA and plotted on the graph on the right; similar results were observed in another independent experiment. (G) Total RNA (~50 µg) extracted from L. major (LV39) at the indicated number of passages after isolation from mouse lesions, were subjected to Northern blot analysis as in (A); graph on the right shows quantification of TERRA signal as in (F); similar results were observed in three independent experiments.

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Fig. 2
: transcription of UUAGGGn-containing RNAs (TERRA) from Trypanosoma brucei telomeres. (A) Total RNA (~15 µg) from procyclic form (PC) (29.13 cell line) and bloodstream form (BS) (SM cell line) of T. brucei were subjected to Northern blot analysis as in Fig. 1A; rRNA (MetBlue) refers to visualisation of rRNA after transfer to the membrane and staining with methylene blue and serves as a loading control. (B) Total RNA from BS form of T. brucei was fractionated on an oligo(dT) resin (Oligotex mRNA Mini Kit, QIAGEN); equivalent volumes from Input, flow through (FT), washes and eluted material were analysed by Northern blot analysis; the material was probed for a-tubulin as a positive control for the binding of poly(A)+ RNA. (C) Total RNA (~15 µg) from T. brucei BS forms was left untreated (Ctl) or subjected to PNK treatment before incubation with TE enzyme; the membrane was probed for a-tubulin as a control for 5’CAP-containing RNA. rRNA(MetBlue) serves as control for 5’ mono-phosphorylated RNAs. (D) Left panel: Northern blot analysis of total RNA (~15 µg) from T. brucei PC and BS forms; rRNA (MetBlue) refers to visualisation of rRNA after transfer to the membrane and staining with methylene blue and serves as a loading control; right panel, total RNA (~15 µg) from T. brucei PC forms was left untreated or treated with DNase I and then subject to Northern blot analysis; the same membrane was probed for a-tubulin as a loading control; DNaseI activity was confirmed using plasmid DNA as substrate, under the same reaction conditions. (E) Total RNA (~15 µg) from T. brucei PC forms was treated as in C and then subjected to Northern blot analysis. (F) T. brucei BS form cells were incubated in medium containing citrate/cis-aconitate to induce the differentiation into PC forms as previously described (Ziegelbauer et al. 1990); total RNA was extracted at the indicated time points after the induction of differentiation and subjected to Northern blot analysis.

Fig. 1 : transcription of UUAGGGn-containing RNAs (TERRA) from Leishmania telomeres. All the experiments in the figure were performed using RNA from promastigote forms. (A) Total RNA (~30 µg) from L. major (Lm; cell lines LT252 and LV39), L. amazonensis (La) and L. braziliensis (Lb) was subjected to Northern blotting and probed with 32P-end labelled (CCCTAA)15 or (TTAGGG)15 oligonucleotides. rRNA ethidium bromide (EtBr) indicates ribosomal RNAs stained with EtBr before transferring to the membrane and serves as loading and quality control of total RNA. (B) Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect TERRA derived from different L. major telomeres; left panel, the diagram indicates the strategy used: the reverse transcription step (RT) was performed with (CCCTAA)15 oligonucleotide (dashed arrow); the cDNA generated was used in a PCR reaction performed with oligonucleotides (arrowheads) specific for the ORF (white arrow) closest to the telomere ((TTAGGG)n); ORFs orientated from the internal portion of the chromosome towards telomeres are indicated as (+); ORFs orientated from the telomeres towards the internal region of the chromosome are indicated as (-); right panel shows EtBr-staining of PCR products after fractionation in agarose gels; the cDNAs used as template for the PCR were generated in reverse transcription reactions performed in the presence (RT+) or absence (RT-) of Reverse Transcriptase; the specific primers for the PCR were designed for ORFs LmjF01.0010, LmjF18.0010, LmjF04.0010, LmjF11.0010, LmjF20.1620 and LmjF23.0030. (C) Left scheme indicates the approach for detection of TERRA by RT-PCR; total RNA (3 µg) from Trypanosoma brucei (Tb; bloodstream form, SM cell line) and L. braziliensis (Lb) was treated with DNAseI and submitted to reverse transcription reaction using oligo(dT) as primer; RT+ and RT- indicates addition or omission of reverse transcriptase enzyme from the cDNA synthesis reaction; the cDNA generated was used as template in a PCR reaction with oligo(dT) and a specific telomere sequence (AGGGTT)4 as primers; right panel shows Ethidium Bromide-staining of PCR products; in the bottom, actin and HTBF genes were used as positive controls for polyadenylated RNA in T. brucei and L. braziliensis, respectively. (D) Total RNA from L. braziliensis was fractionated on an oligo(dT) resin (Oligotex mRNA Mini Kit, QIAGEN); equivalent volumes from Input, flow through (FT), washes and eluted material were analysed by Northern blotting as in (A); samples were probed for a-tubulin as a positive control for the binding of poly(A)+ RNA. (E) Total RNA (~15 µg) from L. braziliensis was left untreated (Ctl) or subjected to polynucleotide kinase treatment (PNK) before incubation with terminator exonuclease (TE); samples were subjected to Northern blot analysis as in (A) and probed for a-tubulin as a control for 5’ CAP-containing RNA; rRNA (MetBlue) refers to rRNA staining with methylene blue after transfer to the membrane and serves as control for 5’mono-phosphorylated RNA. (F) Metacyclic and procyclic parasites were purified as previously described (Sacks et al. 1985). Briefly, stationary-phase L. major (LV39) promastigotes were incubated with the lectin PNA and agglutinated cells (PNA+) were separated from metacyclic parasites (PNA-) by centrifugation; the purification yield was 2.0 to 3.5%; the typical metacyclic morphology was used to confirm the sample purity; total RNA (~50 µg) from PNA+ and PNA- cells were subjected to Northern blot analysis as in (A); TERRA signal was quantified with ImageJ software, normalised with rRNA and plotted on the graph on the right; similar results were observed in another independent experiment. (G) Total RNA (~50 µg) extracted from L. major (LV39) at the indicated number of passages after isolation from mouse lesions, were subjected to Northern blot analysis as in (A); graph on the right shows quantification of TERRA signal as in (F); similar results were observed in three independent experiments.

Fig. 2 : transcription of UUAGGGn-containing RNAs (TERRA) from Trypanosoma brucei telomeres. (A) Total RNA (~15 µg) from procyclic form (PC) (29.13 cell line) and bloodstream form (BS) (SM cell line) of T. brucei were subjected to Northern blot analysis as in Fig. 1A; rRNA (MetBlue) refers to visualisation of rRNA after transfer to the membrane and staining with methylene blue and serves as a loading control. (B) Total RNA from BS form of T. brucei was fractionated on an oligo(dT) resin (Oligotex mRNA Mini Kit, QIAGEN); equivalent volumes from Input, flow through (FT), washes and eluted material were analysed by Northern blot analysis; the material was probed for a-tubulin as a positive control for the binding of poly(A)+ RNA. (C) Total RNA (~15 µg) from T. brucei BS forms was left untreated (Ctl) or subjected to PNK treatment before incubation with TE enzyme; the membrane was probed for a-tubulin as a control for 5’CAP-containing RNA. rRNA(MetBlue) serves as control for 5’ mono-phosphorylated RNAs. (D) Left panel: Northern blot analysis of total RNA (~15 µg) from T. brucei PC and BS forms; rRNA (MetBlue) refers to visualisation of rRNA after transfer to the membrane and staining with methylene blue and serves as a loading control; right panel, total RNA (~15 µg) from T. brucei PC forms was left untreated or treated with DNase I and then subject to Northern blot analysis; the same membrane was probed for a-tubulin as a loading control; DNaseI activity was confirmed using plasmid DNA as substrate, under the same reaction conditions. (E) Total RNA (~15 µg) from T. brucei PC forms was treated as in C and then subjected to Northern blot analysis. (F) T. brucei BS form cells were incubated in medium containing citrate/cis-aconitate to induce the differentiation into PC forms as previously described (Ziegelbauer et al. 1990); total RNA was extracted at the indicated time points after the induction of differentiation and subjected to Northern blot analysis.

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Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids

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[1] + Corresponding author: luiztosi@fmrp.usp.br