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Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging.

Lambertz U, Oviedo Ovando ME, Vasconcelos EJ, Unrau PJ, Myler PJ, Reiner NE - BMC Genomics (2015)

Bottom Line: In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype.We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA.These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada. ulambertz@gmail.com.

ABSTRACT

Background: Leishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis.

Results: We purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes.

Conclusions: These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.

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tRNA-derived fragments are cargo of leishmania exosomes. A. Length distributions of reads mapping to tRNAs in L. donovani and L. braziliensis exosome RNA sequencing libraries. B. Bar graph showing percentages of reads from L. donovani (white bars) and L. braziliensis (grey bars) mapping to the respective tRNA isoacceptors. C. tRNA secondary structures for leishmania tRNA-Asp and tRNA-Leu (downloaded from [38]). Arrowheads indicate major cleavage products as observed in the sequenced libraries. D. Northern blots with probes designed against tRNA-Asp (Asp1) and tRNA-Leu (Leu1 and Leu2). L. donovani total (T) and exosome (E) RNA were probed on the same membrane. Equal amounts of RNA (3 μg) were loaded in each lane. Asp1 and Leu1 probes were designed to specifically detect full length tRNA as well as t-RNA-derived small RNAs seen in sequencing libraries, whereas Leu2 was designed against the mid region (anticodon loop) of tRNA-Leu and hence only detects the full length tRNA.
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Fig5: tRNA-derived fragments are cargo of leishmania exosomes. A. Length distributions of reads mapping to tRNAs in L. donovani and L. braziliensis exosome RNA sequencing libraries. B. Bar graph showing percentages of reads from L. donovani (white bars) and L. braziliensis (grey bars) mapping to the respective tRNA isoacceptors. C. tRNA secondary structures for leishmania tRNA-Asp and tRNA-Leu (downloaded from [38]). Arrowheads indicate major cleavage products as observed in the sequenced libraries. D. Northern blots with probes designed against tRNA-Asp (Asp1) and tRNA-Leu (Leu1 and Leu2). L. donovani total (T) and exosome (E) RNA were probed on the same membrane. Equal amounts of RNA (3 μg) were loaded in each lane. Asp1 and Leu1 probes were designed to specifically detect full length tRNA as well as t-RNA-derived small RNAs seen in sequencing libraries, whereas Leu2 was designed against the mid region (anticodon loop) of tRNA-Leu and hence only detects the full length tRNA.

Mentions: Remarkably, we found a large number of reads in both the L. donovani and L. braziliensis exosome RNA libraries that mapped to tRNA genes. A few recent studies characterizing the RNA content of mammalian exosomes had reported the presence of tRNAs or their fragments in these vesicles. For example, reads mapping to tRNAs were found in sequencing libraries made with RNA from exosomes released from neuronal cells (13.5%) [35], immune cells (~7%) [36] and plasma exosomes (1.24%) [37]. Strikingly, in our datasets, 351,919 reads (36.4 %) and 135,149 reads (21.1%) from L. donovani and L. braziliensis, respectively, mapped to tRNA genes when aligned to the Leishmania major MHOM/IL/81/Friedlin (LmjF) reference genome (which has the best curation on tRNA annotation amongst leishmania species). These frequencies exceeded by some measure those reported for mammalian exosomes in the studies cited above. Close inspection of the genome alignments revealed that a high percentage of these sequences were covering only parts of the respective tRNA genes (Figure 5), consistent with the occurrence of tRNA-derived small RNAs (tsRNAs), which has recently been recognized as a specific process. In light of these findings we decided to characterize the reads mapping to tRNAs in more detail. In case of both libraries, the vast majority (99.8%) of reads were in the sense direction of transcription. Looking at their length profiles, we found the mean read length to be slightly different between the two libraries, 38 nt for L. donovani and 46 nt for L. braziliensis, however, the median read length was similar (33 nt and 34 nt, respectively) (Figure 5A). For both leishmania libraries, tsRNAs derived from tRNA-Asp, tRNA-Gln, tRNA-Glu and tRNA-Leu were most abundantly present (Figure 5B and Table 4). To make a case that these tsRNAs were specific cleavage products that are selectively packaged into exosomes and not just a by-product of tRNA turnover that is disposed by the cell, we calculated the Pearson’s correlation of the predicted cellular amino acid usage and the relative expression of our tsRNAs as assessed by sequencing. The results showed that there was no correlation (r = 0.163 for L. donovani and r = 0.114 for L. braziliensis), indicating that the tsRNAs were unlikely to be random degradation products. Strikingly, we observed the same rank order frequency of tRNA isotypes as origins of tsRNAs in both libraries (Figure 5B and Table 4), indicating that the formation of specific tsRNAs and their appearance as exosomal cargo is an evolutionary conserved phenomenon in leishmania.Figure 5


Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging.

Lambertz U, Oviedo Ovando ME, Vasconcelos EJ, Unrau PJ, Myler PJ, Reiner NE - BMC Genomics (2015)

tRNA-derived fragments are cargo of leishmania exosomes. A. Length distributions of reads mapping to tRNAs in L. donovani and L. braziliensis exosome RNA sequencing libraries. B. Bar graph showing percentages of reads from L. donovani (white bars) and L. braziliensis (grey bars) mapping to the respective tRNA isoacceptors. C. tRNA secondary structures for leishmania tRNA-Asp and tRNA-Leu (downloaded from [38]). Arrowheads indicate major cleavage products as observed in the sequenced libraries. D. Northern blots with probes designed against tRNA-Asp (Asp1) and tRNA-Leu (Leu1 and Leu2). L. donovani total (T) and exosome (E) RNA were probed on the same membrane. Equal amounts of RNA (3 μg) were loaded in each lane. Asp1 and Leu1 probes were designed to specifically detect full length tRNA as well as t-RNA-derived small RNAs seen in sequencing libraries, whereas Leu2 was designed against the mid region (anticodon loop) of tRNA-Leu and hence only detects the full length tRNA.
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Related In: Results  -  Collection

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Fig5: tRNA-derived fragments are cargo of leishmania exosomes. A. Length distributions of reads mapping to tRNAs in L. donovani and L. braziliensis exosome RNA sequencing libraries. B. Bar graph showing percentages of reads from L. donovani (white bars) and L. braziliensis (grey bars) mapping to the respective tRNA isoacceptors. C. tRNA secondary structures for leishmania tRNA-Asp and tRNA-Leu (downloaded from [38]). Arrowheads indicate major cleavage products as observed in the sequenced libraries. D. Northern blots with probes designed against tRNA-Asp (Asp1) and tRNA-Leu (Leu1 and Leu2). L. donovani total (T) and exosome (E) RNA were probed on the same membrane. Equal amounts of RNA (3 μg) were loaded in each lane. Asp1 and Leu1 probes were designed to specifically detect full length tRNA as well as t-RNA-derived small RNAs seen in sequencing libraries, whereas Leu2 was designed against the mid region (anticodon loop) of tRNA-Leu and hence only detects the full length tRNA.
Mentions: Remarkably, we found a large number of reads in both the L. donovani and L. braziliensis exosome RNA libraries that mapped to tRNA genes. A few recent studies characterizing the RNA content of mammalian exosomes had reported the presence of tRNAs or their fragments in these vesicles. For example, reads mapping to tRNAs were found in sequencing libraries made with RNA from exosomes released from neuronal cells (13.5%) [35], immune cells (~7%) [36] and plasma exosomes (1.24%) [37]. Strikingly, in our datasets, 351,919 reads (36.4 %) and 135,149 reads (21.1%) from L. donovani and L. braziliensis, respectively, mapped to tRNA genes when aligned to the Leishmania major MHOM/IL/81/Friedlin (LmjF) reference genome (which has the best curation on tRNA annotation amongst leishmania species). These frequencies exceeded by some measure those reported for mammalian exosomes in the studies cited above. Close inspection of the genome alignments revealed that a high percentage of these sequences were covering only parts of the respective tRNA genes (Figure 5), consistent with the occurrence of tRNA-derived small RNAs (tsRNAs), which has recently been recognized as a specific process. In light of these findings we decided to characterize the reads mapping to tRNAs in more detail. In case of both libraries, the vast majority (99.8%) of reads were in the sense direction of transcription. Looking at their length profiles, we found the mean read length to be slightly different between the two libraries, 38 nt for L. donovani and 46 nt for L. braziliensis, however, the median read length was similar (33 nt and 34 nt, respectively) (Figure 5A). For both leishmania libraries, tsRNAs derived from tRNA-Asp, tRNA-Gln, tRNA-Glu and tRNA-Leu were most abundantly present (Figure 5B and Table 4). To make a case that these tsRNAs were specific cleavage products that are selectively packaged into exosomes and not just a by-product of tRNA turnover that is disposed by the cell, we calculated the Pearson’s correlation of the predicted cellular amino acid usage and the relative expression of our tsRNAs as assessed by sequencing. The results showed that there was no correlation (r = 0.163 for L. donovani and r = 0.114 for L. braziliensis), indicating that the tsRNAs were unlikely to be random degradation products. Strikingly, we observed the same rank order frequency of tRNA isotypes as origins of tsRNAs in both libraries (Figure 5B and Table 4), indicating that the formation of specific tsRNAs and their appearance as exosomal cargo is an evolutionary conserved phenomenon in leishmania.Figure 5

Bottom Line: In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype.We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA.These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada. ulambertz@gmail.com.

ABSTRACT

Background: Leishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis.

Results: We purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes.

Conclusions: These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.

Show MeSH
Related in: MedlinePlus