Donna J. Koslowsky

The insect-phase gRNA transcriptome in Trypanosoma brucei

One of the most striking examples of small RNA regulation of gene expression is the process of RNA editing in the mitochondria of trypanosomes. In these parasites, RNA editing involves extensive uridylate insertions and deletions within most of the mitochondrial messenger RNAs (mRNAs). Over 1200 small guide RNAs (gRNAs) are predicted to be responsible for directing the sequence changes that create start and stop codons, correct frameshifts and for many of the mRNAs generate most of the open reading frame. In addition, alternative editing creates the opportunity for unprecedented protein diversity. In Trypanosoma brucei, the vast majority of gRNAs are transcribed from minicircles, which are approximately one kilobase in size, and encode between three and four gRNAs. The large number (5000–10 000) and their concatenated structure make them difficult to sequence. To identify the complete set of gRNAs necessary for mRNA editing in T. brucei, we used Illumina deep sequencing of purified gRNAs from the procyclic stage. We report a near complete set of gRNAs needed to direct the editing of the mRNAs.

An Intragenic Guide RNA Location Suggests a Complex Mechanism for Mitochondrial Gene Expression in Trypanosoma brucei

ABSTRACT In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5′ end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5′-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5′-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent KD in the 5–10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistent with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events.

Unusual organization of a developmentally regulated mitochondrial RNA polymerase (TBMTRNAP) gene in Trypanosoma brucei

We report here the characterization of a developmentally regulated mitochondrial RNA polymerase transcript in the parasitic protozoan, Trypanosoma brucei. The 3822 bp protein-coding region of the T. brucei mitochondrial RNA polymerase (TBMTRNAP) gene is predicted to encode a 1274 amino acid polypeptide, the carboxyl-terminal domain of which exhibits 29-37% identity with the mitochondrial RNA polymerases from other organisms in the molecular databases. Interestingly, the TBMTRNAP mRNA is one of several mature mRNA species post-transcriptionally processed from a stable, polycistronic precursor. Alternative polyadenylation of the TBMTRNAP mRNA produces two mature transcripts that differ by 500 nt and that show stage-specific differences in abundance during the T. brucei life cycle. This alternative polyadenylation event appears to be accompanied by the alternative splicing of a high abundance, non-coding downstream transcript of unknown function. Our finding that the TBMTRNAP gene is transcribed into two distinct mRNAs subject to differential regulation during the T. brucei life cycle suggests that mitochondrial differentiation might be achieved in part through the regulated expression of this gene.

A Historical Perspective on RNA Editing

The known examples of RNA editing now encompass a variety of alterations of RNA primary sequence that arise from base modifications, nucleotide insertions or deletions, and nucleotide replacements. Hence, the definition of RNA editing has evolved as new systems have been described. This chapter presents a historical perspective on some of the pivotal discoveries that helped direct the current avenues of research in the field of RNA editing.

Cytoplasmic incompatibility following somatic cell fusion in Griffithsia pacifica Kylin, a red alga

SummarySomatic cell fusion between two isolates ofG. pacifica is followed by a cytoplasmic incompatibility reaction (CIR) in the cytoplasm donated by only one of the isolates. This CIR is characterized by the aggregation, fusion and lysis of chloroplasts of the sensitive strain; the chloroplasts of the other strain are unaffected. In addition, the nuclei of both strains retain a normal distribution during the fusion and lysis events. Cell elongation and nuclear division stop in CIR-affected cells. The CIR begins in the hybrid cell and then appears sequentially in adjacent cells of the sensitive strain; this transfer occurs only between living cells which share a crosswall. There is a lag between hybrid cell formation and the initiation of the CIR. This lag is more than 3 times as long at 17 ‡C than at 24 ‡C; over this range, the rate of movement of the CIR along a filament is temperature-insensitive. Thus it appears that a temperature dependent process, perhaps the synthesis of CIR-inducing agents, is required for the initiation of the CIR; subsequent movement of such agents appears to occur by diffusion.

The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5′ end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different “sets” of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a “bank” of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins.

Ultrastructure of selective chloroplast destruction after somatic cell fusion in Griffithsia pacifica Kylin (Rhodophyta)

Fusion of somatic cells from different geographic isolates of Griffithsia pacifica is followed by a cytoplasmic incompatibility reaction (CIR)5 in the cytoplasm donated by only one of the isolates. This CIR is characterized by the aggregation, fusion, and lysis of chloroplasts of the sensitive strain; the cholorplasts of the other strain are unaffected. Within a given cell, chloroplast fusions result in a 50% decrease in chloroplast number in the first 8 h; over the next 3–5 weeks, chloroplast lysis leads to the complete loss of photosynthetic pigments (18). Results of this ultrastructural study indicated that the primary effect of the cytoplasmic incompatibility reaction was a directed destruction of the sensitive strain chloroplasts. No aggregation or abnormalities were seen in organelles other than the chloroplasts. Chloroplast destruction was accompanied by extensive vesiculation of the cytoplasm and a definit change in the physiological state of the cell; the dictyosomes became inactive and there was a reduction in the amount of endoplasmic reticulum, cytoplasmic ribosomes and starch grains. This selective destruction of one chloroplast population in the presence of a second population of unaffected chloroplasts suggests that the incompatibility reaction observed in G. pacifica results from competitive interactions between the two chloroplast types.

Complex interactions in the regulation of trypanosome mitochondrial gene expression

Trypanosomes undergo extreme physiological changes to adapt to different environments as they cycle between hosts. Adaptation to the different environments has evolved an energy metabolism involving a mitochondrion with an unusual genome. Recently, Aphasizhev and colleagues have identified two new protein complexes, a mitochondrial polyadenylation complex and a guide RNA stabilization complex, that provide novel insights into the coordinated expression of the mitochondrial genome.