The polyadenylation complex of<i>Trypanosoma brucei:</i>Characterization of the functional poly(A) polymerase

Koch, H; Raabe, Marco; Urlaub, Henning; Bindereif, Albrecht; Preußer, Christian

doi:10.1080/15476286.2015.1130208

Cited by 40 publications

(49 citation statements)

References 44 publications

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“…Mechanistically trans-splicing is related to the removal of introns in other eukaryotes by cis-splicing, with each gene being in effect an exon. Less is understood about polyadenylation of kinetoplastid mRNAs (96,97), but the reaction follows trans-splicing and localisation of the polyA tail is guided by a polypyrimidine tract and AG sequence upstream of ORFs that predominantly dictate splicing (98,99). DRIP-seq reveals abundant R-loops within the intergenic regions associated with trans-splicing and polyadenylation, with a strong correlation between R-loop enrichment and polyadenylation site density and proximity to the gene CDS.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

Briggs

Hamilton

Crouch

et al. 2018

Preprint

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R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere homeostasis, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked transsplicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localisation was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the intergenic regions of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination, most T. brucei R-loops act in a novel role, promoting RNA Pol II movement or mRNA processing. Finally, we show there is little evidence for correlation between R-loop localisation and mapped sites of DNA replication initiation.

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Section: Discussionmentioning

confidence: 99%

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

Briggs

Hamilton

Crouch

et al. 2018

Preprint

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“…KKIP8 is one of 2 canonical poly(A) polymerases in trypanosomes, PAP1 and PAP2. PAP1 depletion has no detectable effect on mRNA polyadenylation, but causes an elevation of long non-coding RNA levels and precursors of small nucleolar RNAs (Chikne et al, 2017;Koch et al, 2016) KKIP12/RBP34 is a predicted RNA-binding protein that interacts with the trypanosomal homologue of Mkt1p in yeast 2-hybrid screens (Singh et al, 2014).…”

Section: New Components Suggest a Link Between Kinetochore Function Amentioning

confidence: 99%

Ndc80/Nuf2-like protein KKIP1 connects a stable kinetoplastid outer kinetochore complex to the inner kinetochore and responds to metaphase tension

Brusini¹,

D’Archivio²,

McDonald³

et al. 2019

Preprint

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Kinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of its universal function, the composition of kinetochores in extant eukaryotes can differ greatly, and understanding how these different systems evolved and now function are important questions in cell biology. In trypanosomes and other Kinetoplastida, the kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via the inner kinetochore protein KKT4.However, we do not know the extent of the trypanosome kinetochore and proteins interacting with a highly divergent Ndc80/Nuf2-like protein, KKIP1, suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. Two of these core components were recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The complex is physically and biochemically distinct from KKT proteins, but KKIP1 links the inner and outer sets, with its C-terminus very close to the centromere and N-terminus at the outer kinetochore. Moreover, trypanosome kinetochores exhibit intrakinetochore movement during metaphase, primarily by elongation of KKIP1, consistent with pulling at the outer kinetochores. Together, these data suggest that the KOK complex, KKIP5 and N-terminus of KKIP1 likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore.

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“…As noted above, after 1h AN7973 treatment, the level of SLRNA was either unchanged or slightly increased. RNAi targeting splicing or polyadenylation factors has similar effects [39,56,57]. For example, RNAi that targeted polyadenylation factors caused 2-4 fold increases in SLRNA [39].…”

Section: An7973 Inhibits Mrna Processingmentioning

confidence: 99%

“…Trans splicing is spatially and mechanistically coupled to polyadenylation of the preceding mRNA. Polyadenylation sites are dictated by the positions of trans-splicing sites [34][35][36][37], RNAimediated depletion of polyadenylation factors inhibits trans splicing [38,39], and disruption of splicing stops polyadenylation [34][35][36][37]. The spliced leader precursor RNA (SLRNA) is 140 nt long and is synthesised by RNA polymerase II [40] from approximately 200 tandemly repeated genes [41].…”

Section: Introductionmentioning

confidence: 99%

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Begolo

Vincent

Giordani

et al. 2018

Preprint

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Kinetoplastid parasites -trypanosomes and leishmanias -infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. Their targets in trypanosomes were hitherto unknown. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate and reduced levels of mRNA, and accumulation of peri-nuclear granules which are typical for splicing inhibition. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, these results indicate that mRNA processing is the primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing. The EC 50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action, and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition, and resistance after CPSF3 expression did not, however, always correlate, and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. This suggests that the modes of action of oxaboroles that target trypanosome mRNA processing may extend beyond CPSF3 inhibition.

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The polyadenylation complex ofTrypanosoma brucei:Characterization of the functional poly(A) polymerase

Cited by 40 publications

References 44 publications

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

Ndc80/Nuf2-like protein KKIP1 connects a stable kinetoplastid outer kinetochore complex to the inner kinetochore and responds to metaphase tension

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

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