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RNA recognition motif (RRM) containing proteins are important regulators of gene expression in trypanosomes. Here we expand our current knowledge on the exclusively nuclear localized RRM domain containing protein RBP33 of Trypanosoma brucei. Overexpression of RBP33 leads to a quick growth arrest in G2/M in bloodstream form cells likely due to an overall mRNA- and spliced leader abundance decrease while the ribosomal RNAs remain unaffected. The recombinant RBP33 binds to poly(A) and random sequence RNA in vitro confirming its role as a RNA binding protein. Finally super-resolution microscopy detects RBP33 in small punctae throughout the nucleus and surrounding the nucleolus, however the signal is depleted inside the nucleolus., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, an illness that affects 6-7 million people and for which there is no effective drug therapy or vaccine. The publication of its complete genome sequence allowed a rapid advance in molecular studies including in silico screening of genes involved with pathogenicity as well as molecular targets for the development of new diagnostic methods, drug therapies and prophylactic vaccines. Alongside with in silico genomic analyses, methods to study gene function in this parasite such as gene deletion, overexpression, mutant complementation and reporter gene expression have been largely explored. More recently, the use of genome-wide strategies is producing a shift towards a global perspective on gene function studies, with the examination of the expression and biological roles of gene networks in different stages of the parasite life cycle and under different contexts of host parasite interactions. Here we describe the molecular tools and protocols currently available to perform genetic manipulation of the T. cruzi genome, with emphasis on recently described strategies of gene editing that will facilitate large-scale functional genomic analyses. These new methodologies are long overdue, since more efficient protocols for genetic manipulation in T. cruzi are urgently needed for a better understanding of the biology of this parasite and molecular processes involved with the complex and often harmful, interaction with its human host., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we make use of existing Trypanosoma brucei ribosome profiling data to provide evidence of ribosome occupancy (and likely translation) of mRNAs from 225 currently unannotated coding sequences (CDSs). A small number of these putative genes correspond to extra copies of previously annotated genes, but 85% are novel. The median size of these novels CDSs is small (81 aa), indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs confirmed here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as protein-coding genes. Nonetheless, approximately one-third of the new CDSs were found only in T. brucei subspecies. Using ribosome footprints, RNA-Seq and spliced leader mapping data, we updated previous work to definitively revise the start sites for 414 CDSs as compared to the current gene models. The data pointed to several regions of the genome that had sequence errors that altered coding region boundaries. Finally, we consolidated this data with our previous work to propose elimination of 683 putative genes as protein-coding and arrive at a view of the translatome of slender bloodstream and procyclic culture form T. brucei., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ∼39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Previous studies of Brugia malayi promoters have suggested that they are unusual in that they lack the CAAT or TATAA boxes that are often emblematic of eucaryotic core promoter domains. Instead, the region surrounding the spliced leader (SL) addition site appears to function as the core promoter domain in B. malayi. To test the hypothesis that polymorphisms in this SL addition domain are important determinants of promoter activity, a series of domain swap mutants were prepared replacing the SL addition domain of the B. malayi 13kDa large subunit ribosomal protein (BmRPL13) with those of other ribosomal protein (RP) promoters exhibiting a wide range of activities. These constructs were then tested for promoter activity in a homologous transient transfection system. On average, polymorphisms in the SL addition domain were found to be responsible for 80% of the variation in promoter activity exhibited by the RP promoters tested. Essentially all of this effect could be attributable to polymorphisms in the 10nt located directly upstream of the SL addition site. A comparison of the sequence of this domain to the promoter activity exhibited by the domain swap mutants suggested that promoter activity was related to the number of T residues present in the coding strand of the upstream domain. Confirming this, mutation of the upstream domain of the promoter of the BmRPS4 gene to a homogeneous stretch of 10 T residues resulted in a significant increase in promoter activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Spliced leader trans-splicing adds a short exon, the spliced leader (SL), to pre-mRNAs to generate 5' ends of mRNAs. Addition of the SL in metazoa also adds a new cap to the mRNA, a trimethylguanosine (m(3)(2,2,7)GpppN) (TMG) that replaces the typical eukaryotic monomethylguanosine (m7GpppN)(m7G) cap. Both trans-spliced (m3(2,2,7)GpppN-SL-RNA) and not trans-spliced (m7GpppN-RNA) mRNAs are present in the same cells. Previous studies using cell-free systems to compare the overall translation of trans-spliced versus non-trans-spliced RNAs led to different conclusions. Here, we examine the contribution of m3(2,2,7)GpppG-cap and SL sequence and other RNA elements to in vivo mRNA translation and stability in nematode embryos. Although 70-90% of all nematode mRNAs have a TMG-cap, the TMG cap does not support translation as well as an m7G-cap. However, when the TMG cap and SL are present together, they synergistically interact and translation is enhanced, indicating both trans-spliced elements are necessary to promote efficient translation. The SL by itself does not act as a cap-independent enhancer of translation. The poly(A)-tail synergistically interacts with the mRNA cap enhancing translation and plays a greater role in facilitating translation of TMG-SL mRNAs. In general, recipient mRNA sequences between the SL and AUG and the 3' UTR do not significantly contribute to the translation of trans-spliced mRNAs. Overall, the combination of TMG cap and SL contribute to mRNA translation and stability in a manner typical of a eukaryotic m7G-cap and 5' UTRs, but they do not differentially enhance mRNA translation or stability compared to RNAs without the trans-spliced elements.

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5' splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5' end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5' splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5'-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes.

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions -34 to -31 had a detrimental effect on transcription in vitro: the CC dinucleotide overlaps one element necessary in vivo. Similarly. four nucleotides (TGAC; positions -67 to -64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the -60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.

In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli, showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.

In the DNA of African trypanosomes a small fraction of thymine is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J). The function of this large base is unknown. The presence of J in the silent variant surface glycoprotein gene expression sites and the lack of J in the transcribed expression site indicates that DNA modification might play a role in control of gene repression. However, the abundance of J in the long telomeric repeat tracts and in subtelomeric arrays of simple repeats suggests that J may also have specific functions in repetitive DNA. We have now analyzed chromosome-internal repetitive sequences in the genome of Trypanosoma brucei and found J in the minichromosomal 177-bp repeats, in the long arrays of 5S RNA gene repeats, and in the spliced-leader RNA gene repeats. No J was found in the rDNA locus or in dispersed repetitive transposon-like elements. Remarkably, the rDNA of T. brucei is not organized in long arrays of tandem repeats, as in many other eukaryotes. T. brucei contains only approximately 15-20 rDNA repeat units that are divided over six to seven chromosomes. Our results show that J is present in many tandemly repeated sequences, either at a telomere or chromosome internal. The presence of J might help to stabilize the long arrays of repeats in the genome.

We have assessed the potential of using the spliced leader (SL) or mini-exon gene as a marker for molecular phylogenetic analysis of genus Trypanosoma. A total of 27 trypanosome sequences were compared, 18 of these being newly reported. In contrast to genus Leishmania, we found the non-transcribed spacer region of the SL locus in trypanosomes to be far too variable for informative comparison of all but the most closely related species. At the other extreme, the short (39 nt) SL exon was usually completely conserved and hence uninformative. The SL RNA showed variation in both length (97-152 nt) and sequence among different trypanosome species, with most variation occurring in stem-loop II. Consequently, this region could not be aligned with confidence in multiple sequence alignment, severely reducing the number of phylogenetically informative nucleotide positions. In computer simulation, most of the SL RNAs readily folded into the 3 stem-loop secondary structure predicted previously, but again stem-loop II was highly variable. No obvious correlation could be seen between the length of this stem-loop and trypanosome biology. We conclude that the SL repeat is not an informative phylogenetic marker for long range evolutionary studies of genus Trypanosoma.

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5' terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at -39 to -33 and a GACN5G element at -66 to -58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.