Analysis of the Trypanosoma brucei EATRO 164 Bloodstream Guide RNA Transcriptome

Abstract

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.

Transcription initiation defines kinetoplast RNA boundaries

“…The mitochondrion undergoes dramatic changes in function, size, and gene expression during the digenetic life cycle of T. brucei. The developmental variations in abundance, the 3′ modification state, and the extent of editing have been documented for most mitochondrial mRNAs (34), but few of these factors correlate with expected requirements for a specific protein at a particular life-cycle stage. Major advances in understanding mRNA editing, polyadenylation, and translation processes (31,35) have left the decades-old notion of unregulated multicistronic transcription unperturbed.…”

Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization

“…The maxicircles contain 18 protein-coding genes, 12 of which require modification by U insertion/deletion RNA editing to generate their open reading frames (ORFs), and these are thus referred to as cryptogenes ( 2 – 5 ). The minicircles encode small non-coding RNAs, including the 50–70 bp guide RNAs (gRNAs) that serve as trans-acting templates to direct RNA editing ( 4 , 6 – 8 ). mRNAs encoded by nine of the cryptogenes are edited throughout their lengths and require the utilization of multiple gRNAs, and these are termed ‘pan-edited’ (reviewed in ( 9 ) and ( 5 )).…”

Trypanosomatid mitochondrial RNA editing: dramatically complex transcript repertoires revealed with a dedicated mapping tool

“…This process repeats until editing is completed; therefore, the editing process is generally 3′ to 5′ directional. Hundreds of gRNAs are needed to ‘decrypt’ all mitochondrial transcripts, and kinetoplastid genomes retain far more gRNAs than the minimum required to execute editing ( 19 – 21 ).…”

“…It is also possible that multiple RNAs coding for different protein sequences could be generated from a single cryptogene ( 29 – 31 ). Deep sequencing of gRNAs ( 19 , 20 , 32 ) and targeted deep sequencing of editing intermediates ( 12 , 18 , 24 ) have revealed both non-productive editing events and potentially functional alternative editing.…”