Regulation of gene expression in trypanosomatids: living with polycistronic transcription

Clayton, Christine

doi:10.1098/rsob.190072

Cited by 206 publications

(250 citation statements)

References 314 publications

(516 reference statements)

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“…First, we tested for the existence of specific sequence motifs associated with the distinct patterns of DNA synthesis initiation around these sites. As expected, MEME analysis retrieved motifs that have been associated with transcription initiation and termination, known to take place at these sites (G/T rich motifs and polypyrimidine tracks; Fig.S3) (31). We could not find any sequence signature that correlated specifically with sites of DNA synthesis initiation, or that differed between the regions used during ES phase or throughout the cell cycle.…”

Section: Distinct Strand Asymmetry and G4 Distribution At Sites Of Cosupporting

confidence: 59%

“…Notably, virtually all RNA polymerase (pol) II transcribed genes in Leishmania (24) and T. brucei (25) have been shown to be transcribed as multigene transcription units, each of which possesses a single poorly defined transcription start site that appears to be constitutively active (26). The ubiquitous use of multigenic transcription is likely to be a universal feature of kinetoplastids (27) and means that gene expression is perhaps exclusively regulated at the post-transcriptional level (28)(29)(30)(31). Two further consequences arise from multigene transcription.…”

Section: Introductionmentioning

confidence: 99%

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Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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Once every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase.Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex.Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite. sequence read depth in the former relative to the latter. As result, DNA synthesis very late in S-phase, or that occurs outside of S-phase, would escape detection. To test this, we modified the MFA-seq approach in order that DNA content enrichment could be calculated in replicating cells relative to naturally occurring non-replicative cells. Mapping origins of replication by calculating DNA enrichment in exponentially growing cells versus cells in a stationary state has been used successfully in bacteria (42) and yeast (43). Thus, we reasoned that L. major in stationary phase could also serve as a non-replicative control for normalization in MFA-seq analysis compared with cells that are growing exponentially. To test this, we used flow cytometry to compare DNA content of exponentially growing and stationary phase cells and, in addition, compared the capacity of the two cell populations to incorporate the thymidine analogue 5-ethynyl-2'deoxyuridine (EdU; Fig. 1A,B). Flow cytometry showed that the proportion of cells in S phase (with DNA content between 1C and 2C) was substantially lower in a population of stationary cells compared with exponentially growing cells (Fig. 1A). Concomitantly, stationary phase cells, unlike exponentially growing cells, failed to incorporate EdU, even upon relatively long periods of incubation ( Fig. 1B). Thus, L. major cells in stationary phase do not perform any detectable DNA synthesis and were therefore deemed suitable to be used as the non-replicative sample in MFA-seq analysis.Next, we compared MFA-seq profiles using read depth ratios from FACS-sorted early S (ES) and G2 cells, and from exponentially growing cells (EXP) and stationary (STA) cells (Fig. 1C). As reported ...

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Section: Distinct Strand Asymmetry and G4 Distribution At Sites Of Cosupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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“…As in other Kinetoplastids, transcription in T. brucei is polycistronic, and mRNAs are excised by co-transcriptional trans splicing and polyadenylation. RNA binding proteins play important roles throughout the lifetime of every mRNA, governing mRNA processing, translation, and mRNA decay (Clayton, 2019, Kolev et al, 2014.…”

Section: Introductionmentioning

confidence: 99%

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-formTrypanosoma brucei

Liu

Marucha

Clayton

2019

Preprint

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KeywordsmRNA Trypanosoma translation binding RNASeq Summary ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x) 8 C(x) 5 C(x) 3 H zinc finger motifs. ZC3H20 is unstable in mammalian-infective bloodstream forms, but becomes more abundant as they transform to growth-arrested stumpy form, while ZC3H21 appears only in the procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least seventy of the bound mRNAs decrease after RNAi targeting ZC3H20 or ZC3H20 and ZC3H21; their products include procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes procyclic forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.the fates of several hundred mRNAs, and have overlapping but different roles in the trypanosome life cycle and in gene regulation.

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“…In Trypanosoma brucei, most regulation of gene expression is post-transcriptional. Proteincoding genes are arranged in polycistronic transcription units, and mRNAs are excised by trans splicing and polyadenylation [1]. Levels of constitutively expressed proteins and mRNAs are strongly influenced by codon usage [2,3], while regulation during development, over the cell cycle, and in response to environmental conditions is effected mainly by RNAbinding proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Levels of constitutively expressed proteins and mRNAs are strongly influenced by codon usage [2,3], while regulation during development, over the cell cycle, and in response to environmental conditions is effected mainly by RNAbinding proteins. The latter often, but not always, bind sequences in 3'-untranslated regions (3'-UTRs) [1]. All mRNAs -whether or not they are subject to specific regulation -are expected to be bound by numerous different proteins, forming a "messenger ribonucleoprotein" (mRNP) assembly.…”

Section: Introductionmentioning

confidence: 99%

The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-formTrypanosoma brucei

Bajak

Leiss

Clayton

et al. 2019

Preprint

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Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

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Regulation of gene expression in trypanosomatids: living with polycistronic transcription

Cited by 206 publications

References 314 publications

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-formTrypanosoma brucei

The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-formTrypanosoma brucei

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