Current Genomics - Volume 19, Issue 2, 2018

From synthesis to decay, mRNA associates with RNA-binding proteins (RBPs) establishing dynamic ribonucleoprotein particles (RNPs). Understanding the composition and function of RNPs is fundamental to understanding how eukaryotic mRNAs are controlled. This is especially relevant for trypanosomes and related kinetoplastid parasites, which mostly rely on posttranscriptional mechanisms to control gene expression. Crucial for trypanosome differentiation, development, or even response to heat shock, RBPs are known to be essential modulators of diverse molecular processes. The recent application of large-scale quantitative methods, such as Next- Generation Sequencing (NGS) and quantitative mass spectrometry, has revealed new exciting features about the parasite RNA-related metabolism. Novel proteins carrying RNA-binding activity, including many proteins without RNA-related ontology were discovered setting a necessary groundwork to get in insights into RNA biology. Conclusion: This review aims to give the reader an understanding of current trypanosome RNP research, highlighting the progress made using high-throughput approaches.

Trypanosomatids are a group of kinetoplastid parasites including some of great public health importance, causing debilitating and life-long lasting diseases that affect more than 24 million people worldwide. Among the trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the Leishmania genus are the most well studied parasites, due to their high prevalence in human infections. These parasites have an extreme genomic and phenotypic variability, with a massive expansion in the copy number of species-specific multigene families enrolled in host-parasite interactions that mediate cellular invasion and immune evasion processes. As most trypanosomatids are heteroxenous, and therefore their lifecycles involve the transition between different hosts, these parasites have developed several strategies to ensure a rapid adaptation to changing environments. Among these strategies, a rapid shift in the repertoire of expressed genes, genetic variability and genome plasticity are key mechanisms. Trypanosomatid genomes are organized into large directional gene clusters that are transcribed polycistronically, where genes derived from the same polycistron may have very distinct mRNA levels. This particular mode of transcription implies that the control of gene expression operates mainly at post-transcriptional level. In this sense, gene duplications/losses were already associated with changes in mRNA levels in these parasites. Gene duplications also allow the generation of sequence variability, as the newly formed copy can diverge without loss of function of the original copy. Recently, aneuploidies have been shown to occur in several Leishmania species and T. cruzi strains. Although aneuploidies are usually associated with debilitating phenotypes in superior eukaryotes, recent data shows that it could also provide increased fitness in stress conditions and generate drug resistance in unicellular eukaryotes. In this review, we will focus on gene and chromosomal copy number variations and their relevance to the evolution of trypanosomatid parasites.

Introduction: Understanding how the nuclear genome of kinetoplastid parasites is replicated received experimental stimulus from sequencing of the Leishmania major, Trypanosoma brucei and Trypanosoma cruzi genomes around 10 years ago. Gene annotations suggested key players in DNA replication initiation could not be found in these organisms, despite considerable conservation amongst characterised eukaryotes. Initial studies that indicated trypanosomatids might possess an archaeal- like Origin Recognition Complex (ORC), composed of only a single factor termed ORC1/CDC6, have been supplanted by the more recent identification of an ORC in T. brucei. However, the constituent subunits of T. brucei ORC are highly diverged relative to other eukaryotic ORCs and the activity of the complex appears subject to novel, positive regulation. The availability of whole genome sequences has also allowed the deployment of genome-wide strategies to map DNA replication dynamics, to date in T. brucei and Leishmania. ORC1/CDC6 binding and function in T. brucei displays pronounced overlap with the unconventional organisation of gene expression in the genome. Moreover, mapping of sites of replication initiation suggests pronounced differences in replication dynamics in Leishmania relative to T. brucei. Conclusion: Here we discuss what implications these emerging data may have for parasite and eukaryotic biology of DNA replication.

Introduction: An important portion of the Trypanosoma cruzi genome is composed of mobile genetic elements, which are interspersed with genes on all chromosomes. The L1Tc non-LTR retrotransposon and its truncated version NARTc are the most highly represented and best studied of these elements. L1Tc is actively transcribed in all three forms of the Trypanosoma parasite and encodes the proteins that enable it to autonomously mobilize. This mini review discusses the enzymatic properties of L1Tc that enable its mobilization and possibly the mobilization of other non-autonomous retrotransposons in Trypanosoma. We also briefly review the Hepatitis Delta Virus-like autocatalytic and 2A self-cleaving viral-like sequences contained in L1Tc that regulate post-transcriptional properties such as relative protein abundance and mRNA stability. Special emphasis is placed on the Pr77 dual system, which is based on the RNA pol II-dependent internal promoter of L1Tc and NARTc and the HDV-like ribozyme activity encoded by the first 77 nucleotides of the element's DNA and RNA. The high degree of conservation of the Pr77 sequence, referred to as the “Pr77-hallmark”, among different trypanosomatid retroelements suggests that these mobile elements are responsible for the distribution of regulatory sequences within the genome they inhabit. Conclusion: We also discuss how the involvement of L1Tc and NARTc in the gene regulatory processes of these parasites could justify their domestication and long-term coexistence in these ancient organisms.

Introduction: Trypanosoma brucei uses antigenic variation to evade the host antibody clearance by periodically changing its Variant Surface Glycoprotein (VSGs) coat. T. brucei encode over 2,500 VSG genes and pseudogenes, however they transcribe only one VSG gene at time from one of the 20 telomeric Expression Sites (ESs). VSGs are transcribed in a monoallelic fashion by RNA polymerase I from an extranucleolar site named ES body. VSG antigenic switching occurs by transcriptional switching between telomeric ESs or by recombination of the VSG gene expressed. VSG expression is developmentally regulated and its transcription is controlled by epigenetic mechanisms and influenced by a telomere position effect. Conclusion: Here, we discuss 1) the molecular basis underlying transcription of telomeric ESs and VSG antigenic switching; 2) the current knowledge of VSG monoallelic expression; 3) the role of inositol phosphate pathway in the regulation of VSG expression and switching; and 4) the developmental regulation of Pol I transcription of procyclin and VSG genes.

Introduction: Phylogenetic relationships between different lineages of Trypanosoma cruzi, the agent of Chagas disease, have been controversial for several years. However, recent phylogenetic and phylogenomic analyses clarified the nuclear relationships among such lineages. However, incongruence between nuclear and kinetoplast DNA phylogenies has emerged as a new challenge. This incongruence implies several events of mitochondrial introgression at evolutionary level. However, the mechanism that gave origin to introgressed lineages is unknown. Here, I will review and discuss how maxicircles of the kinetoplast were horizontally and vertically transferred between different lineages of T. cruzi. Conclusion: Finally, I will discuss what we know - and what we don't - about the kDNA transference and inheritance in the context of sexual reproduction in this parasite.

The Trypanosomatid family includes flagellated parasites that cause fatal human diseases. Remarkably, protein-coding genes in these organisms are positioned in long tandem arrays that are transcribed polycistronically. However, the knowledge about regulation of transcription initiation and termination in trypanosomatids is scarce. The importance of epigenetic regulation in these processes has become evident in the last years, as distinctive histone modifications and histone variants have been found in transcription initiation and termination regions. Moreover, multiple chromatin-related proteins have been identified and characterized in trypanosomatids, including histone-modifying enzymes, effector complexes, chromatin-remodelling enzymes and histone chaperones. Notably, base J, a modified thymine residue present in the nuclear DNA of trypanosomatids, has been implicated in transcriptional regulation. Here we review the current knowledge on epigenetic control of transcription by all three RNA polymerases in this group of early-diverged eukaryotes.

Background: Leptomonas pyrrhocoris is a parasite of the firebug Pyrrhocoris apterus. This flagellate has been recently proposed as a model species for studying different aspects of the biology of monoxenous trypanosomatids, including host – parasite interactions. During its life cycle L. pyrrhocoris never tightly attaches to the epithelium of the insect gut. In contrast, its dixenous relatives (Leishmania spp.) establish a stable infection via attachment to the intestinal walls of their insect hosts. Material and Methods: This process is mediated by chemical modifications of the cell surface lipophosphoglycans. In our study we tested whether the inability of L. pyrrhocoris to attach to the firebug's midgut is associated with the absence of these glycoconjugates. We also analyzed evolution of the proteins involved in proper lipophosphoglycan assembly, cell attachment and establishment of a stable infection in L. pyrrhocoris, L. seymouri, and Leishmania spp. Our comparative analysis demonstrated differences in SCG/L/R repertoire between the two parasite subgenera, Leishmania and Viannia, which may be related to distinct life strategies in various Leishmania spp. The genome of L. pyrrhocoris encodes 6 SCG genes, all of which are quite divergent from their orthologs in the genus Leishmania. Using direct probing with an antibody recognizing the β-Gal side chains of lipophosphoglycans, we confirmed that these structures are not synthesized in L. pyrrhocoris. Conclusion: We conclude that either the SCG enzymes are not active in this species (similarly to SCG5/7 in L. major), or they possess a different biochemical activity.