Guide RNA-binding complex from mitochondria of trypanosomatids.

In the mitochondria of trypanosomatids, the majority of mRNAs undergo massive uracil-insertion/deletion editing. Throughout the processes of pre-mRNA polyadenylation, guide RNA (gRNA) uridylylation and annealing to mRNA, and editing reactions, several multiprotein complexes must engage in transient interactions to produce a template for protein synthesis. Here, we report the identification of a protein complex essential for gRNA stability. The gRNA-binding complex (GRBC) interacts with gRNA processing, editing, and polyadenylation machineries and with the mitochondrial edited mRNA stability (MERS1) factor. RNAi knockdown of the core subunits, GRBC1 and GRBC2, led to the elimination of gRNAs, thus inhibiting mRNA editing. Inhibition of MERS1 expression selectively abrogated edited mRNAs. Homologous proteins unique to the order of Kinetoplastida, GRBC1 and GRBC2, form a stable 200 kDa particle that directly binds gRNAs. Systematic analysis of RNA-mediated and RNA-independent interactions involving the GRBC and MERS1 suggests a unified model for RNA processing in the kinetoplast mitochondria.

3' adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria.

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post-transcriptionally and requires extensive U-insertion/deletion mRNA editing. In mitochondrial extracts, 3' adenylation reportedly influences degradation kinetics of synthetic edited and pre-edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A-tails typically found in mitochondrial mRNAs, and decreases the abundance of never-edited and edited transcripts. Pre-edited mRNAs are not destabilized by the lack of 3' adenylation, whereas short A-tails are required and sufficient to maintain the steady-state levels of partially edited, fully edited, and never-edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A-tail in edited molecules. Upon completion of the editing process, the short A-tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3' processing and editing of mitochondrial mRNAs in trypanosomes.

Disruption of Toxoplasma gondii-Induced Host Cell DNA Replication Is Dependent on Contact Inhibition and Host Cell Type.

The protozoan Toxoplasma gondii is a highly successful obligate intracellular parasite that, upon invasion of its host cell, releases an array of host-modulating protein effectors to counter host defenses and further its own replication and dissemination. Early studies investigating the impact of T. gondii infection on host cell function revealed that this parasite can force normally quiescent cells to activate their cell cycle program. Prior reports by two independent groups identified the dense granule protein effector HCE1/TEEGR as being solely responsible for driving host cell transcriptional changes through its direct interaction with the cyclin E regulatory complex DP1 and associated transcription factors. Our group independently identified HCE1/TEEGR through the presence of distinct repeated regions found in a number of host nuclear targeted parasite effectors and verified its central role in initiating host cell cycle changes. Additionally, we report here the time-resolved kinetics of host cell cycle transition in response to HCE1/TEEGR, using the fluorescence ubiquitination cell cycle indicator reporter line (FUCCI), and reveal the existence of a block in S-phase progression and host DNA synthesis in several cell lines commonly used in the study of T. gondii. Importantly, we have observed that this S-phase block is not due to additional dense granule effectors but rather is dependent on the host cell line background and contact inhibition status of the host monolayer in vitro. This work highlights intriguing differences in the host response to reprogramming by the parasite and raises interesting questions regarding how parasite effectors differentially manipulate the host cell depending on the in vitro or in vivo context. IMPORTANCE Toxoplasma gondii chronically infects approximately one-third of the global population and can produce severe pathology in immunologically immature or compromised individuals. During infection, this parasite releases numerous host-targeted effector proteins that can dramatically alter the expression of a variety of host genes. A better understanding of parasite effectors and their host targets has the potential to not only provide ways to control infection but also inform us about our own basic biology. One host pathway that has been known to be altered by T. gondii infection is the cell cycle, and prior reports have identified a parasite effector, known as HCE1/TEEGR, as being responsible. In this report, we further our understanding of the kinetics of cell cycle transition induced by this effector and show that the capacity of HCE1/TEEGR to induce host cell DNA synthesis is dependent on both the cell type and the status of contact inhibition.

Identification and characterization of nuclear non-canonical poly(A) polymerases from Trypanosoma brucei.

Regulation of nuclear genome expression in Trypanosoma brucei is critical for this protozoan parasite's successful transition between its vertebrate and invertebrate host environments. The canonical eukaryotic circuits such as modulation of transcription initiation, mRNA splicing and polyadenylation appear to be nearly non-existent in T. brucei suggesting that the transcriptome is primarily defined by mRNA turnover. Our previous work has highlighted sequence similarities between terminal RNA uridylyl transferases (TUTases) and non-canonical poly(A) polymerases, which are widely implicated in regulating nuclear, cytoplasmic and organellar RNA decay throughout the eukaryotic lineage. Here, we have continued characterization of TUTase-like proteins in T. brucei and identified two nuclear non-canonical poly(A) polymerases (ncPAPs). The 82kDa TbncPAP1 is essential for viability of procyclic and bloodstream forms of T. brucei. Similar to Trf4/5 proteins from budding yeast, TbncPAP1 requires protein cofactor(s) to exert poly(A) polymerase activity in vitro. The recombinant 54kDa TbncPAP2 showed a PAP activity as an individual polypeptide. Proteomic analysis of the TbncPAP1 interactions demonstrated its association with Mtr4 RNA helicase and several RNA binding proteins, including a potential ortholog of Air1p/2p proteins, which indicates the presence of a stable TRAMP-like complex in trypanosomes. Our findings suggest that TbncPAP1 may be a "quality control" nuclear PAP involved in targeting aberrant or anti-sense transcripts for degradation by the 3'-exosome. Such mechanisms are likely to play a major role in alleviating promiscuity of the transcriptional machinery.

Toxoplasma Effector Recruits the Mi-2/NuRD Complex to Repress STAT1 Transcription and Block IFN-γ-Dependent Gene Expression.

Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses.

The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice.

Polymorphic rhoptry-secreted kinases (ROPs) are essential virulence factors of Toxoplasma gondii. In particular, the pseudokinase ROP5 is the major determinant of acute virulence in mice, but the underlying mechanisms are unclear. We developed a tandem affinity protein tagging and purification approach in T. gondii and used it to show that ROP5 complexes with the active kinases ROP18 and ROP17. Biochemical analyses indicate that ROP18 and ROP17 have evolved to target adjacent and essential threonine residues in switch region I of immunity-related guanosine triphosphatases (GTPases) (IRGs), a family of host defense molecules that function to control intracellular pathogens. The combined activities of ROP17 and ROP18 contribute to avoidance of IRG recruitment to the intracellular T. gondii-containing vacuole, thus protecting the parasite from clearance in interferon-activated macrophages. These studies reveal an intricate, multilayered parasite survival strategy involving pseudokinases that regulate multiple active kinase complexes to synergistically thwart innate immunity.