Identification and stage-specific association with the translational apparatus of TbZFP3, a CCCH protein that promotes trypanosome life-cycle development

The post-transcriptional control of gene expression is becoming increasingly important in the understanding of regulated events in eukaryotic cells. The parasitic kinetoplastids have a unique reliance on such processes, because their genome is organized into polycistronic transcription units in which adjacent genes are not coordinately regulated. Indeed, the number of RNA-binding proteins predicted to be encoded in the genome of kinetoplastids is unusually large, invoking the presence of unique RNA regulators dedicated to gene expression in these evolutionarily ancient organisms. Here, we report that a small CCCH zinc finger protein, TbZFP3, enhances development between life-cycle stages in Trypanosoma brucei. Moreover, we demonstrate that this protein interacts both with the translational machinery and with other small CCCH proteins previously implicated in trypanosome developmental control. Antibodies to this protein also co-immunoprecipitate EP procyclin mRNA and encode the major surface antigen of insect forms of T. brucei. Strikingly, although TbZFP3 is constitutively expressed, it exhibits developmentally regulated association with polyribosomes, and mutational analysis demonstrates that this association is essential for the expression of phenotype. TbZFP3 is therefore a novel regulator of developmental events in kinetoplastids that acts at the level of the post-transcriptional control of gene expression

Antigenic variation in <i>Trypanosoma brucei</i>: joining the DOTs

African trypanosomes, such as &lt;i&gt;Trypanosoma brucei&lt;/i&gt;, are protistan parasites that cause sleeping sickness. Though first described more than a century ago, trypanosomes remain a blight on the health of the human population and on the economy of sub-Saharan Africa. &lt;i&gt;T. brucei&lt;/i&gt; replicates in the bloodstream of infected mammals and traverses the blood-brain barrier to enter the central nervous system in the late, frequently fatal, stages of the disease. Because of its extracellular lifestyle, &lt;i&gt;T. brucei&lt;/i&gt; is continuously exposed to antibody challenge. To circumvent this, the parasite uses antigenic variation of a surface protein named the variant surface glycoprotein (VSG). Around 107 VSG molecules are expressed on the parasite's cell surface, creating a dense coat that prevents adaptive immunity from detecting or accessing invariant antigens. However, antibodies against the expressed VSG are generated, and periodic switches to an immunologically distinct VSG coat are necessary for parasite survival. Such switches are pre-emptive of the immune response and contribute to the pattern of trypanosome growth seen in an infected host (Figure 1): parasite numbers increase, but then drop as VSG-specific antibodies are raised by the host. Cells that have switched to another VSG coat survive this killing and seed the outgrowth of a subsequent peak of parasites, which is again decimated by anti-VSG immune killing. As a survival strategy, antigenic variation succeeds by prolonging the time that the parasite

The role of genomic location and flanking 3'UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I-transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16-mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I-transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell-cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell-cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I-transcribed ES, as well as conserved VSG 3′UTR 16-mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

Extracellular parasites of the mammalian bloodstream face considerable challenges including incessant assault by the immune system. African trypanosomes are consummate survivors in this inclement environment and are renowned for their supremely sophisticated strategy of antigenic variation of their protective surface coat during the course of chronic infections. Recent developments are making us realize how complex this antigenic machinery is and are allowing us to tackle previously intractable problems. However, many of the simplest (and arguably the most important) questions still remain unanswered

Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements

Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain. Components of this complex represent a form of post-transcriptional operon because trypanosome mitochondrial activity is unusual in being developmentally programmed. By genome analysis we identified the genes for seven components of the COX complex. Each mRNA exhibits bloodstream stage-specific instability, which is not mediated by the RNA silencing pathway but which is alleviated by cycloheximide. Reporter assays have identified regulatory regions within the 3′-untranslated regions of three COX mRNAs operating principally at the translational level, but also via mRNA stability. Interrogation of the mapped regions via oligonucleotide frequency scoring provides evidence for genome-wide conservation of regulatory sequences among a large cohort of procyclic-enriched transcripts. Analysis of the co-regulated subunits of a stage-specific enzyme is therefore a novel approach to uncover cryptic regulatory sequences controlling gene expression at the post-transcriptional level

Architecture of the trypanosome RNA editing accessory complex, MRB1

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation

NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control

Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1

The 5′-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m3G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m2,2,7G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m7GTP and the reaction product S-adenosyl-l-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, α-helical domain of 40 amino acids, which is essential for m7G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1

RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs

The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5′- and 3′-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and ‘RNA walk’ to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite’s life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes

Depletion of Trypanosome CTR9 Leads to Gene Expression Defects

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1