A major line of our research is to understand the organization, function, and expression of the molecules present in the surface coat of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. The surface glycoprotein coat of this parasite is composed of members of two large families: mucins that are involved in protection and cell invasion, and trans-sialidases that incorporate the sugar sialic acid from host glycoconjugates into parasite mucins. T. cruzi has two hosts, a blood-sucking insect (Triatomina) and a mammalian host such as humans. Furthermore, the gene families from which mucins and trans-sialidases are expressed differ depending on whether the parasite is in the insect or mammalian host stage. Each family is composed of dozens to hundreds of members that are likely to be expressed simultaneously in a given stage of the parasite.
One of the main tasks of the parasite is to coordinate the stage-specific expression of this large number of mucin and trans-sialidase genes in each of its hosts. Regulation of expression in these cells cannot be accomplished at the transcriptional level because transcription in trypanosomes is polycistronic. Containing from tens to hundreds of cistrons, these long transcripts are processed into mature transcripts through trans-splicing at the 5′-end and polyadenylation at the 3′-end. Given that the cistrons present in a polycistron encode proteins with unrelated functions, trypanosomatids have to make use of post-transcriptional mechanisms to effectively control gene expression. Thus, once polycistrons are processed, the resulting mature mRNAs might be translated or degraded according to environmental conditions and the parasite's needs.
One mechanism of post-transcriptional regulation we analyzed in some detail in trypanosomatids is the modulation of mRNA stability. This process involves defined motifs in the 3′ untranslated region (3′ UTR) of a transcript and RNA-binding proteins recognizing these specific motifs. Thus, it is the composition of such ribonucleoprotein complexes that modulates gene expression at the post-transcriptional level, determining the fate of the transcript—either translation or degradation. We have identified a number of motifs in the 3′ UTR of mucin transcripts, motifs whose deletion modifies the mRNA half-life and translation efficiency. Likewise, we found that a family of RNA-binding proteins characterized by the presence of RNA recognition motifs (RRM) was expressed in trypanosomatids; some were shown to modulate mucin transcript stability. RRM-containing proteins have, in addition to the RRM interacting with the target mRNA, domains likely to be involved in protein-protein interactions, allowing the proteins to incorporate other proteins into the ribonucleoprotein complexes. The mechanisms modulating mRNA stability/translation efficiency are similar to those occurring in other eukaryotic cells; we believe that the major difference may be that in trypanosomatids, these events occur in most or even all transcripts.
Recently, we explored the possibility that other, parasite-specific mechanisms might modulate gene expression in these cells. We obtained evidence that some intercistronic regions are not trans-spliced/polyadenylated during polycistronic unit processing. The lack of processing generates dicistronic units; we identified a dicistronic unit that contains the coding sequences for the RRM-containing proteins TcUBP1 and TcUBP2 (T. cruzi uridine-binding proteins 1 and 2). In contrast to the monocistronic units, this dicistron has a long half-life (hours versus minutes). In addition, a second trans-splicing/polyadenylation site that is skipped during the processing of the corresponding polycistron is located in the 3′ UTR of the transcripts encoding the trans-sialidase. As a consequence, the mRNAs encoding this enzyme contain elongated 3′ UTRs, which are further processed by trans-splicing and polyadenylation. Preliminary evidence suggests that, before translation, processing of both dicistronic units and the 3′ UTR of the trans-sialidase transcripts has to be accomplished by trans-splicing. Given that the cistrons involved are differentially expressed during the developmental stages of the parasite in the insect and mammalian hosts, we posit that trans-splicing is required to regulate gene expression. To test our hypotheses, we plan to analyze transcript processing and how this affects expression/degradation of the mature transcript.
Also, we plan to identify RNA-binding proteins that recognize intercistronic regions and prevent trans-splicing/polyadenylation events. Our results suggest that one protein that binds to the intercistronic region is PTB (polypyrimidine-tract binding protein). PTB is known to be involved in RNA metabolism, including alternative splicing in higher eukaryotic cells. The possibility that this protein is involved in skipping of the trans-splicing polyadenylation sites is therefore worth analyzing. Lack of processing of transcripts might be a way to maintain a pool of pre-mature RNAs in readiness for translation. Indeed, we have observed that trypansosomes contain cytoplasmic bodies (P-body–like), which feature mRNAs and RNA-binding proteins. Further work will indicate whether precursor/mature RNAs, in the form of ribonucleoprotein complexes, are stored in such cytoplasmic bodies. Given that the parasite lacks transcriptional regulation of gene expression, storage of mRNA or premature transcripts for translation might be an interesting alternative way for the parasite to rapidly adapt to different environments (insect versus mammal).
Last updated July 2010