A comprehensive understanding of host-pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing 'dual RNA-seq', in which gene expression changes in both the pathogen and the host are analysed simultaneously.

/pdf/dual-rna-seq-of-pathogen-and-host-1dk955su2j.pdf

Dual RNA-seq of pathogen and host

The identification of RNAs that are not translated into proteins was an important breakthrough, defining the diversity of molecules involved in eukaryotic regulation of gene expression. These non-coding RNAs can be divided into two main classes according to their length: short non-coding RNAs, such as microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). The lncRNAs in association with other molecules can coordinate several physiological processes and their dysfunction may impact in several pathologies, including cancer and infectious diseases. They can control the flux of genetic information, such as chromosome structure modulation, transcription, splicing, messenger RNA (mRNA) stability, mRNA availability, and post-translational modifications. Long non-coding RNAs present interaction domains for DNA, mRNAs, miRNAs, and proteins, depending on both sequence and secondary structure. The advent of new generation sequencing has provided evidences of putative lncRNAs existence; however, the analysis of transcriptomes for their functional characterization remains a challenge. Here, we review some important aspects of lncRNA biology, focusing on their role as regulatory elements in gene expression modulation during physiological and disease processes, with implications in host and pathogens physiology, and their role in immune response modulation.

https://www.mdpi.com/2311-553X/5/1/17/pdf

Long Non-Coding RNAs in the Regulation of Gene Expression: Physiology and Disease

Argonaute proteins are conserved throughout all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participate in host defense by DNA interference, whereas eukaryotic Argonaute proteins (eAgos) control a wide range of processes by RNA interference. Here we review molecular mechanisms of guide and target binding by Argonaute proteins, and describe how the conformational changes induced by target binding lead to target cleavage. On the basis of structural comparisons and phylogenetic analyses of pAgos and eAgos, we reconstruct the evolutionary journey of the Argonaute proteins through the three domains of life and discuss how different structural features of pAgos and eAgos relate to their distinct physiological roles.

/pdf/the-evolutionary-journey-of-argonaute-proteins-1ck6h82t9q.pdf

The evolutionary journey of Argonaute proteins

Disease characteristics and serological responses in patients with differing severity of COVID-19 infection: A longitudinal cohort study in Dhaka, Bangladesh Afroza Akter1☯‡, Tasnuva Ahmed1☯‡, Imam Tauheed, Marjahan Akhtar, Sadia Isfat Ara RahmanID , Fatema Khaton, Faisal Ahmmed, Jannatul FerdousID , Mokibul Hassan AfradID , Zannat Kawser, Mohabbat Hossain, Rabeya KhondakerID , Mohammad Abul Hasnat, Mostafa Aziz SumonID , Asif RashedID , Shuvro Ghosh, Stephen B. Calderwood, Richelle C. Charles, Edward T. Ryan, Purvesh KhatriID , Holden Terry MaeckerID , Gerlinde Obermoser, Bali Pulendran, John D. ClemensID , Sayera Banu, Tahmina ShirinID , Regina C. LaRocque, Jason B. HarrisID , Taufiqur Rahman Bhuiyan, Fahima Chowdhury, Firdausi QadriID *

PLOS Neglected Tropical Diseases

https://www.cell.com/cell/pdf/S0092-8674(14)00143-3.pdf

Discovery of Unconventional Kinetochores in Kinetoplastids

The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5′ and 3′ ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

/pdf/the-transcriptome-of-the-human-pathogen-trypanosoma-brucei-4r1lz9tj86.pdf

The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

Centrins are Ca(2+)-binding proteins that have been implicated in a number of biological processes, including organelle duplication, mRNA export, DNA repair and signal transduction. In the protozoan parasite Trypanosoma brucei we have previously described TbCentrin2, which is present on a bi-lobed structure, and involved in the duplication and segregation of the Golgi complex. Recently, another centrin, TbCentrin4, was also found at the bi-lobe and has been implicated in organelle segregation and cytokinesis. We now show that cytokinesis is not inhibited, but that a dysregulation of nuclear and cell division leads to the production of zoids - daughter siblings that contain all organelles except the nucleus. Our results, therefore, suggest that TbCentrin4 is involved in processes that coordinate karyokinesis and cytokinesis.

/pdf/centrin4-coordinates-cell-and-nuclear-division-in-t-brucei-2zfk5dvsnp.pdf

Centrin4 coordinates cell and nuclear division in T. brucei.

African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals, have a complex digenetic life cycle between a mammalian host and an insect vector, the blood-feeding tsetse fly. Although the importance of the insect vector to transmit the disease was first realized over a century ago, many aspects of trypanosome development in tsetse have not progressed beyond a morphological analysis, mainly due to considerable challenges to obtain sufficient material for molecular studies. Here, we used high-throughput RNA-Sequencing (RNA-Seq) to profile Trypanosoma brucei transcript levels in three distinct tissues of the tsetse fly, namely the midgut, proventriculus and salivary glands. Consistent with current knowledge and providing a proof of principle, transcripts coding for procyclin isoforms and several components of the cytochrome oxidase complex were highly up-regulated in the midgut transcriptome, whereas transcripts encoding metacyclic VSGs (mVSGs) and the surface coat protein brucei alanine rich protein or BARP were extremely up-regulated in the salivary gland transcriptome. Gene ontology analysis also supported the up-regulation of biological processes such as DNA metabolism and DNA replication in the proventriculus transcriptome and major changes in signal transduction and cyclic nucleotide metabolism in the salivary gland transcriptome. Our data highlight a small repertoire of expressed mVSGs and potential signaling pathways involving receptor-type adenylate cyclases and members of a surface carboxylate transporter family, called PADs (Proteins Associated with Differentiation), to cope with the changing environment, as well as RNA-binding proteins as a possible global regulators of gene expression.

/pdf/transcriptome-profiling-of-trypanosoma-brucei-development-in-ibondtz9gi.pdf

Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans.

At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, Tb AGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs). To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified Tb AGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (Tb DCL1) or nuclear (Tb DCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs. Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself.

/pdf/small-interfering-rna-producing-loci-in-the-ancient-5c2x3zs2q0.pdf

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

Summary
DYF-13, originally identified in Caenorhabditis elegans within a collection of dye-filling chemosensory mutants, is one of several proteins that have been classified as putatively involved in intraflagellar transport (IFT), the bidirectional movement of protein complexes along cilia and flagella and specifically in anterograde IFT. Although genetic studies have highlighted a fundamental role of DYF-13 in nematode sensory cilium and trypanosome flagellum biogenesis, biochemical studies on DYF-13 have lagged behind. Here, we show that in Trypanosoma brucei the orthologue to DYF-13, PIFTC3, participates in a macromolecular complex of approximately 660 kDa. Mass spectroscopy of affinity-purified PIFTC3 revealed several components of IFT complex B as well as orthologues of putative IFT factors DYF-1, DYF-3, DYF-11/Elipsa and IFTA-2. DYF-11 was further analysed and shown to be concentrated near the basal bodies and in the flagellum, and to be required for flagellum elongation. In addition, by coimmunoprecipitation we detected an interaction between DYF-13 and IFT122, a component of IFT complex A, which is required for retrograde transport. Thus, our biochemical analysis supports the model, proposed by genetic analysis in C. elegans, that the trypanosome orthologue of DYF-13 plays a central role in the IFT mechanism.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2010.07322.x

Biochemical analysis of PIFTC3, the Trypanosoma brucei orthologue of nematode DYF‐13, reveals interactions with established and putative intraflagellar transport components

Joseph B. Franklin

Papers

The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

Centrin4 coordinates cell and nuclear division in T. brucei.

Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans.

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

Biochemical analysis of PIFTC3, the Trypanosoma brucei orthologue of nematode DYF‐13, reveals interactions with established and putative intraflagellar transport components