Transcription mechanisms and genetic expression in T cruzi

Unique mechanisms of control of gene expression and gene expression profiling

There are marked differences in the way prokaryotes and eukaryotes regulate their gene expression. Being part of a group of early branching eukaryotes, trypanosoma- tids have attracted the attention of parasitologists not only for their medical relevance but also because they present distinctive features in their mechanisms for controlling gene expression. The absence of defined RNA polymerase II promoter sequences controlling the expression of individual protein-coding genes, the RNA polymerase I-mediated transcription of a select group of genes, and the requirement of pre-mRNA cis-splicing as a RNA processing event are major characteristics of eukaryotic gene transcription.34 In trypanosomatids, transcription is polycistronic, i.e., several genes are transcribed in one large pre-mRNA, and because of the lack of introns, with only four exceptions,9 cis-splicing does not occur in these organisms. However, since primary transcripts are polycistronic, cleavage of the pre- mRNA has to occur in the nucleus in order to produce monocistronic mRNAs that are capped and polyadenylated. In trypanosomes, cleavage of the pre-mRNA is linked to the addition of the 39 nucleotide miniexon (or spliced leader, SL) containing a methylated cap at the 5’ end and the poly (A) tail at the 3’ end of each mRNA.35 mRNA biosynthesis in these organisms is also notable because of the fact that most mitochondrial mRNAs have to undergo extensive RNA editing before mitochondrial proteins can be produced.36

Adaptation of trypanosomes to distinct environments in the vertebrate and invertebrates hosts as well as differentiation in distinct parasite forms, calls for major changes in morphology, surface composition, biochemical pathways, and thus, complex mechanisms to control gene expression. In most eukaryotes, transcriptional regulation is a major step of gene expression control. In trypanosomes, there is a total lack of evidence for differential regulation of RNA polymerase II transcription and no identifiable RNA polymerase II promoter consensus sequence in the genomes. Thus, the lack of transcription initiation control implies that the knowledge of elements involved in posttranscriptional processes, such as trans-splicing, mRNA stabilization, and translation is crucial for the understanding of gene expression in these organisms. As previously indicated, gene organization in trypanosome chromosomes is also very peculiar. Large polycistronic transcription units encoding 20 or more proteins in one strand separated by strand switch regions (i.e., changes of the coding strand) were found initially in the L. major genome37 and later in the Tritryp genomes.10 Before transcription is completed, the long pre-mRNA is processed in the nucleus by cleavage reactions that are coupled to two cotranscriptional RNA-processing events: trans-splicing of a small capped RNA of 39—41 nucleotides, the spliced leader RNA (SL-RNA) which is added to the 50-terminus of all known protein-encoding RNAs, and 30-end polyadenylation. Both events are dependent on polypyrimidine motifs (polyPY) located within the intergenic regions.35 Again, in contrast to most eukaryotes, no canonical polyA addition signal has been identified, and only AG dinucleotides situated downstream from a polyPY motif are used as SL acceptor site.10,38 Since mRNAs derived from the same polycistronic mRNA precursor can present vast differences in their steady state levels, gene expression modulation must depend heavily on regulatory pathways acting at the control of mRNA half-life. It is believed that by employing this type of regulation, trypanosomes can ensure that rapid changes associated with transmission between insect vector and mammalian host are followed by an instant reprogramming of genetic expression.

Most of the early studies on gene expression in trypanosomatids were focused on the process of antigenic variation, the powerful survival strategy devised by African trypanosomes and allowing T. brucei bloodstream forms to escape the immune defenses from the mammalian host. Variant Surface Glycoproteins (VSGs)39 are the main surface molecules present at the surface of T. brucei bloodstream forms. While the genome of this parasite contains about 1000 VSG genes, only one VSG, present in telomeric locations called VSG bloodstream expression site (BES), is active at a time.40 Understanding the mechanisms controlling VSG expression, particularly the in situ switch (i.e., the mechanism responsible for the activation of one telomeric BES concomitantly with the inactivation of all other

~15 VSG BES), has been a difficult task, but has allowed the discovery of a large body of information about gene expression in this group of organisms (for a recent review, see Stockdale et al.41). Notably, transcription of the VSG located in a BES is mediated by an RNA polymerase I that is present in an extranucleolar location identified as expression site body.42 Compared to T. brucei, studies on gene expression in T. cruzi had a late start. Whereas the first T. cruzi gene was cloned in 1986,43 characterization of some of the key players involved in gene expression control in this parasite has only recently begun.

Initial studies on stage-specific gene expression in T. cruzi indicated that, similar to what had already been described for T. brucei and Leishmania, the majority of T. cruzi genes are constitutively transcribed in epimastigotes, trypomastigotes, and amastigotes.44-46 Further studies on a number of gene models showed that change in mRNA stability is a main mechanism employed by T. cruzi to control stage- specific gene expression of protein-coding genes.44-48 From these studies, the 30 UTR has emerged as a main regulatory site involved in controlling mRNA stability. Using transient transfections with CAT or luciferase reporter genes, various groups have demonstrated the presence of elements in the 30 UTR of several mRNAs that confer developmental regulation of the reporter genes.46,49,50 The two examples below illustrate some of these studies. In the T. cruzi genome a tandem array of alternating genes encoding amastin, a surface glycoprotein, and tuzin, a G-like protein, is polycistronically transcribed in all three forms of the parasites’ life cycle. In spite of the constitutive transcription, steady state levels of amastin genes are 60-fold higher in amastigotes compared to epimastigote forms, whereas tuzin mRNA levels do not change significantly. It has been shown that the half-life of amastin mRNAs is sevenfold longer in amastigotes than in epimastigotes and that a 180-nt sequence present in the 30 UTR is responsible for amastin upregula- tion.51 This positive effect is likely mediated by a sequence that binds to a RNA stabilizing factor present in amastigotes.46,51 Mucin genes are part of an even larger family of cell surface proteins of T. cruzi with hypervariable regions and with members of distinct subfamilies expressed in various stages of the parasite life cycle52,53 have shown that mRNAs for one group of mucins, SMUG mucin mRNAs, are more abundant in the insect stage and that the mRNA turnover is controlled by an AU- rich element (ARE) located in their 30-UTR. These authors have also demonstrated that an RNA-binding protein named TcUBP-1 is involved in mRNA destabilization in vivo through binding to the ARE of SMUG mucin mRNAs.54 They have gone further in characterizing this trans-acting factor, showing that TcUBP-1 is part of a ~450 kDa ribonucleoprotein complex with a poly(A)-binding protein and a novel 18kDa RNA-binding protein, named TcUBP-2.55 The two examples above show that both positive and negative regulatory elements controlling mRNA stability are found in the genome of T. cruzi and that these sequences are recognized by transacting factors. Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM).56 It is thus likely that a large number of these proteins are key players in processes controlling pre-mRNA trans-splicing, transport and mRNA decay, but so far, only a few of them have been characterized in T. cruzi.51-59 More recently, the identification of the glucosylated thymine DNA base ((3-D-glucosyl-hydroxymethyluracil) (or base J) in the T. cruzi genome revealed the existence of epigenetic mechanisms controlling transcription in this parasite.60 Base J is also present in silent telomeric repeats in the T. brucei genome as well as in Leishmania where it was found to be essential for proper transcription termination of the polycistronic transcription units.6162

Transitions in gene expression that occur during differentiation of T. cruzi have also been analyzed using high throughput strategies that recently became available. Reports on microarray analyses confirm that it is a valuable screening tool for identifying stage-regulated genes in T. cruzi.63~65 From these studies, we can infer that a total of almost 5000 transcripts (approx. 50% of T. cruzi genes) are regulated during the parasite life cycle, supporting the conclusion that transcript abundance is one of the main levels of gene expression regulation in T. cruzi. Together with more recent studies using next generation cDNA sequencing technologies, these analyses allow researchers to identify groups of genes that are part what has been called “posttranscriptional regulons,” consisting of mRNAs that show almost identical patterns of regulation.66 Using RNA-seq and ribosome profiling, Smircich et al. were able to assess the extent of regulation of the transcriptome and the translatome during differentiation from epimastigotes to the infective metacyclic trypomastigote stage.67 This study showed that translational regulation, in addition to regulation of steady state level of mRNA, is a significant mechanism controlling gene expression in the parasite. Also using RNA-seq, a recent study investigated global transcrip- tome dynamics simultaneously captured in T. cruzi parasite and host cells in an infection time course of human fibroblasts.68 Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. The findings suggested that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, this comprehensive, high resolution transcriptomic dataset provided a substantially more detailed interpretation of T. cruzi infection biology and offered a basis for future drug and vaccine discovery efforts.68

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