The mitochondrial genome of Trypanosoma brucei contains many cryptogenes that must be extensively edited following transcription. The RNA editing process is directed by guide RNAs (gRNAs) that encode the information for the specific insertion and deletion of uridylates required to generate translatable mRNAs. We have deep sequenced the gRNA transcriptome from the bloodstream form of the EATRO 164 cell line. Using conventionally accepted fully edited mRNA sequences, ~1 million gRNAs were identified. In contrast, over 3 million reads were identified in our insect stage gRNA transcriptome. A comparison of the two life cycle transcriptomes show an overall ratio of procyclic to bloodstream gRNA reads of 3. 5: 1. This ratio varies significantly by gene and by gRNA populations within genes. The variation in the abundance of the initiating gRNAs for each gene, however, displays a trend that correlates with the developmental pattern of edited gene expression. A comparison of related major classes from each transcriptome revealed a median value of ten single nucleotide variations per gRNA. Nucleotide variations were much less likely to occur in the consecutive Watson-Crick anchor region, indicating a very strong bias against G: U base pairs in this region. This work indicates that gRNAs are expressed during both life cycle stages, and that differential editing patterns observed for the different mitochondrial mRNA transcripts are not due to the presence or absence of gRNAs. However, the abundance of certain gRNAs may be important in the developmental regulation of RNA editing.
The life cycle of Trypanosoma brucei involves two distinct environments, the animal host and the insect vector. These environments are distinct in temperature and nutrient composition, providing a unique challenge to T. brucei as it cycles between hosts. In the bloodstream, trypanosomes exist in two forms, the actively dividing slender form and the non-dividing stumpy form. The slender form is optimized to utilize its glucose rich environment, using glycolysis to generate energy [1]. The stumpy form appears to be transitional, activating mitochondrial genes in preparation for uptake in a blood meal by its tsetse fly vector and subsequent transfer to a harsher environment [1]. Once inside the tsetse fly, the parasite utilizes proline to drive oxidative phosphorylation and ATP production in the mitochondrion [2]. While the activity of the mitochondrion is relatively low during the bloodstream stage (BS), expression of the mitochondrial genome is still essential [3,4]. In T. brucei, the mitochondrial genome consists of two types of DNA molecules, maxicircles and minicircles. Maxicircles are 22kb circular DNA that contain the genes for two ribosomal RNAs, 12S and 9S, and eighteen mRNA genes [5]. While some of the protein-coding genes do not require RNA editing prior to translation, most require extensive editing before they can be translated [for review see 6,7]. This process involves the insertion of hundreds of uridylates (U) s and less frequently deletion of Us, often doubling the size of the transcript. The sequence changes are guided by small complementary RNA molecules (the guide RNAs) that are encoded on the minicircles [8]. Minicircles make up the bulk of the kinetoplastid network (anywhere from 5,000–10,000 present in each network) with each minicircle encoding 3–5 gRNAs. In T. brucei, there are more than 200 different minicircle sequence classes (~1200 gRNAs) [8]. Distinct differences in mitochondrial transcript abundance, polyadenylation and the extent of RNA editing are observed during the complex life cycle (Table 1). The pattern of differential RNA editing observed is especially interesting. For example, the cytochrome b (CYb) and cytochrome oxidase II (COII) mRNAs are edited during the insect stage, but are primarily unedited in bloodstream forms [9,10]. In contrast, editing of the NADH dehydrogenase subunit transcripts (ND3, ND7, ND8 and ND9) and editing of the ribosomal protein subunit 12 transcript (RPS12) appears to occur preferentially in bloodstream forms [5,11–15]. Other transcripts, cytochrome oxidase III (COIII) and ATPase subunit 6 (A6) are edited in both life cycle stages [16,17]. Early studies using both Northern blot and primer extension analyses on a limited number of gRNAs indicate that gRNAs are present in both insect and bloodstream forms, suggesting that the regulation of RNA editing is not at the level of gRNA availability [13,18,19]. Our lab has previously published deep sequencing results of the gRNA transcriptome of the T. brucei EATRO 164 procyclic form [20]. Here we present the deep sequencing data for the gRNA transcriptome of a bloodstream form of EATRO 164. A total of 211 populations of gRNAs were identified. We define a population as a group of gRNAs that may vary in sequence, but direct the editing of the same or near same region of the mRNA. Because kinetoplastid RNA editing allows G: U base pairing, most populations contain multiple sequence classes that can guide the generation of the same mRNA sequence. While the number of populations identified was similar to the number identified in the procyclic gRNA transcriptome (214 populations), the total number of gRNAs identified was much reduced and the coverage was less complete; full complements of gRNAs were only identified for COIII and CYb. In spite of the reduced number of gRNAs, an interesting correlation was found that suggests a relationship between the relative abundance of initiating gRNAs between stages and the developmental pattern of mRNA editing.
T. brucei brucei clone IsTar from stock EATRO 164 were grown in rats and isolated as previously described [37]. Bloodstream forms were virtually all long-slender forms isolated after 4 days of infection. Parasites were used immediately for isolation of mitochondria using differential centrifugation as previously described or stored frozen at -80°C until RNA extraction [20]. Both total RNA from whole parasites and mitochondrial RNA (mtRNA) from purified mitochondria were isolated by the acid guanidinium-phenol-chloroform method [38]. Rats were raised according to the animal husbandry guidelines established by Michigan State University. All vertebrate animal use procedures were approved by MSU’s Institutional Animal Care and Use Committee (Application 03/11-051-00). MSU has filed with the Office of Laboratory Animal Welfare (OLAW) an assurance document that commits the university to compliance with NIH policy and the Guide for the Care and Use of laboratory Animals. Samples of mtRNA and total RNA were both treated with DNAse RQI and size fractioned on a polyacrylamide gel as previously described [20]. Guide RNAs were extracted from the gel and prepped for sequencing using the Illumina ‘Small RNA’ protocol as previously described [20]. Libraries from both mtRNA and total RNA samples were deep sequenced on Illumina GAIIx. Reads were then processed and trimmed as previously described [20]. Data with two or more Ns, shorter than 20nts after trimming or with an overall mean Q-score < 25 were discarded. Redundant reads were then removed, while maintaining the number of redundant reads and reads containing fewer than 4 consecutive Ts were removed. To identify gRNAs, each transcript read was aligned to the conventionally edited mRNAs based on known base pairing rules (canonical Watson-Crick base pairs and the G-U base pair). In the initial screen, no gaps were allowed in the alignment, allowing the formulation of the gRNA-mRNA alignment as an extended longest common substring (LCS) problem as previously described [10]. Matched gRNAs were then scored (two points for G: C and A: U base pairs and one point for G: U base pairs). gRNAs with scores >45 were identified as guiding a specific region based on the identified mRNA fully edited sequence. Additional searches with reduced stringency (scores >30) were performed on regions with low gRNA coverage. The matched gRNAs were sorted into populations based on their guiding positions, and the populations analyzed and sorted into major sequence classes.
In order to determine if the BS gRNA transcriptome contained a full complement of guide RNAs, the gRNA populations were aligned to the fully edited mRNAs (S2 Fig). We note, that for an mRNA to be fully edited, not only must all editing sites on the mRNA be covered by a gRNA, the downstream gRNA must generate the anchor binding site for the subsequent gRNA. Therefore, adjacent gRNAs must overlap. Overall, there was an average of 17 nts of overlap between adjacent gRNAs, with the average overlap varying slightly by gene (Table 3). As the median Watson-Crick Anchor is 11 nts, in most cases, the overlap extends beyond the Watson-Crick anchor of the subsequent gRNA. However, we did observe a number of regions where the overlap is minimal. Currently, there is no data that stipulates the minimum anchor needed for efficient editing. However, we postulate that similar to microRNAs, for an anchoring sequence to be sufficiently specific, it should be at least six nucleotides [40]. Indeed, when examining the overlaps between most gRNAs, there are only ten (four procyclic and six BS) that are less than six nucleotides (Fig 5). We therefore used six nucleotides as a cut off to identify regions with potential missing guide RNAs for both life cycle stage transcriptomes. In contrast to the procyclic data, where full complements of gRNAs were identified for five of the mRNA transcripts (A6, COIII, CR4, CYb, and RPS12), in the BS transcriptome, a full complement of gRNAs was only identified for COIII and CYb. Overall, there are 12 edited regions where no gRNAs were identified, and five regions with weak gRNA overlaps in the BS data (Table 5). Of these 17 regions, seven belong to ND7 alone. Interestingly, nine of the 17 missing populations are in very low abundance in the procyclic data, having 100 or fewer reads. Because the number of reads in the BS data is ~3. 5 fold less abundant, this could account for some of these regions of poor coverage. There are six regions that lack gRNA coverage in both data sets. These are found in CR3, MurfII, ND3 and ND7 (Table 5). Interestingly, three of these regions are close to the 3’ end of their respective genes. Regions of weak overlap (ND9 (238–242), ND9 (609–612) ) and regions without gRNA coverage (CR3 (278–292), ND8 (541–553) ) that are unique to the procyclic transcriptome were also observed. Interestingly, the regions of poor procyclic coverage are found in CR3, ND8 and ND9, all transcripts that are preferentially edited in the BS form [5,13,14,34]. While the number of reads in the BS data is less abundant than the procyclic data in general, there are 87 gRNA populations with more identified reads than in the procyclic data set (Table 6). These populations are found in every gene except CYb and MurfII, but most of the populations belong to one of the NADH dehydrogenase subunits, particularly, ND7 or ND9. Another trend worth noting is that the all of the genes with fully edited transcripts that are more abundant in the BS stage, (CR3, CR4, ND3, ND7, ND8, and ND9, with the exception of RPS12), have a higher percentage of classes that are more abundant in the BS (Table 6).
Surprisingly, complete gRNA coverage was observed only for the pan-edited COIII and for CYb, where editing is limited to the 5’ end. The identification of the CYb gRNAs was expected, as it has been previously reported that the gRNAs are present in both life cycle stages even though editing of CYb is limited to the procyclic stage [8,9]. The full coverage of COIII was also not surprising, as COIII was shown to be fully edited and equally abundant in both stages [17]. However, we expected to see complete coverage of ATPase 6 and RPS12 as both of these transcripts have been shown to be essential in both life cycle stages [3,44,45,50]. For ATPase 6, we did identify a total of 29 gRNA populations that do cover all of the editing sites. However, one of the gRNAs (bsA6 (643–667) ) has a single nucleotide mismatch (C: U) and one would introduce a frameshift (bsA6 (520–553) ). The C: U mismatch occurs near the middle of the gRNA, placing the C: U mismatch in a region that is unusually high in Gs and Cs (S2A Fig). It may be that the G: C basepairs immediately upstream of the mismatch stabilize the gRNA/mRNA interaction, allowing it to be tolerated. The frameshift gRNA is also interesting, as it occurs just upstream (1 editing site) of another site where we had previously observed a frameshift sequence anomaly. Both frameshifts (the BS 4U and the Procyclic 11U) generate a predicted protein with nearly the same amino acid sequence. As the frameshifts occur downstream of the highly conserved amino acid region involved in proton translocation [16], it may be that this different carboxyl terminus is tolerated. Near full coverage is also observed for RPS12. For this transcript, one BS identified gRNA (bsRPS12 (96–121) ) has an A-nt insertion that disrupts the gRNA complementarity. Surprisingly, the other mRNA transcript found with near complete coverage was ND9 (one gRNA has a single nt mismatch). All of the other mitochondrially encoded Complex I members did have substantial gaps in coverage. Currently, there is considerable debate on the necessity of Complex I subunits for either stage of the trypanosome life cycle. Studies using RNAi and knockout cell lines of nuclear-encoded members of Complex I have shown that the complex is unnecessary for survival in either life cycle stage [51,52]. However, the nuclear-encoded Complex I member genes are maintained [29], and while we not did identify full coverage for the ND transcripts, a vast majority of the gRNAs were found in both life cycle stages. This study used high-throughput sequencing to characterize the gRNA transcriptome during the bloodstream stage of the trypanosome life cycle. This work suggests that gRNAs are expressed during both life cycle stages, and that differential editing patterns observed for the different mitochondrial mRNA transcripts are not due to the presence or absence of gRNAs. SAMN04302078, SAMN04302079, SAMN04302080, and SAMN04302081 NCBI’s Sequence Read Archive.
Trypanosoma brucei is the causative agent of African sleeping sickness, a disease that threatens millions of people in sub-Saharan Africa. During its life cycle, Trypanosoma brucei lives in either its mammalian host or its insect vector. These environments are very different, and the transition between these environments is accompanied by changes in parasite energy metabolism, including distinct changes in mitochondrial gene expression. In trypanosomes, mitochondrial gene expression involves a unique RNA editing process, where U-residues are inserted or deleted to generate the mRNA’s protein code. The editing process is directed by a set of small RNAs called guide RNAs. Our lab has previously deep sequenced the gRNA transcriptome of the insect stage of T. brucei. In this paper, we present the gRNA transcriptome of the bloodstream stage. Our comparison of these two transcriptomes indicates that most gRNAs are present in both life cycle stages, even though utilization of the gRNAs differs greatly during the two life-cycle stages. These data provide unique insight into how RNA systems may allow for rapid adaptation to different environments and energy utilization requirements.
sequencing techniques
messenger rna
parasitic protozoans
parasitology
developmental biology
trypanosoma brucei gambiense
protozoans
genome analysis
energy-producing organelles
mitochondria
bioenergetics
molecular biology techniques
rna sequencing
cellular structures and organelles
research and analysis methods
life cycles
molecular biology
biochemistry
rna
trypanosoma
cell biology
nucleic acids
rna editing
transcriptome analysis
genetics
biology and life sciences
genomics
computational biology
organisms
parasitic life cycles
Analysis of the Trypanosoma brucei EATRO 164 Bloodstream Guide RNA Transcriptome