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Abstract
Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a “double-cut” elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.
Highlights
In the eukaryotic cell division cycle, many proteins involved in the replication of the cell and its components are expressed exactly when required, ensuring tight control over replicative processes and increasing cellular efficiency
We previously identified a small group of mRNAs that are associated with the PUF-domain protein PUF9, and whose levels peak in late S-phase/early G2 [16]
Expression profiling of starvation-synchronized cells Procyclic (PC; insect-form) T. brucei cells were synchronized by starvation and induced to resume the cell cycle by dilution into fresh media as described previously [16,18]
Summary
In the eukaryotic cell division cycle, many proteins involved in the replication of the cell and its components are expressed exactly when required, ensuring tight control over replicative processes and increasing cellular efficiency. Regulation at the level of transcription has been thoroughly documented: for example, in yeast, at least nine transcription factors central to cellcycle regulation operate in a network to control the expression of each other and of downstream effectors of cell cycle progression [1]. Downstream targets of these transcription factors include cyclins, DNA replication proteins and structural proteins such as histones. The kinetoplastids are an early-branching group of unicellular eukaryotes including several important parasitic pathogens of humans and animals Their life-cycles involve alternation between two very different hosts, typically vertebrates and biting insects, each of which represent considerable, but very different, challenges to the parasites’ survival. Kinetoplastids are excellent models for post-transcriptional control of gene expression in eukaryotes
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