Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function.

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal.

Lytic Reactivation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Is Accompanied by Major Nucleolar Alterations.

The nucleolus is a subnuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is available to date.

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T.

A long noncoding RNA promotes parasite differentiation in African trypanosomes.

The parasite causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes.

Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown.

Genome instability drives epistatic adaptation in the human pathogen .

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen , which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment.

Pseudouridines on Trypanosoma brucei mRNAs are developmentally regulated: Implications to mRNA stability and protein binding.

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high-throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7).

Developmentally Regulated Novel Non-coding Anti-sense Regulators of mRNA Translation in .

The parasite is the causative agent of sleeping sickness and cycles between insect and mammalian hosts. The parasite appears to lack conventional transcriptional regulation of protein coding genes, and mRNAs are processed from polycistronic transcripts by the concerted action of splicing and polyadenylation. Regulation of mRNA function is mediated mainly by RNA binding proteins affecting mRNA stability and translation.

The large repertoire of 2'-O-methylation guided by C/D snoRNAs on Trypanosoma brucei rRNA.

The parasite cycles between insect and mammalian hosts, and is the causative agent of sleeping sickness. Here, we performed genome-wide mapping of 2'--methylations (Nms) on trypanosome rRNA using three high-throughput sequencing methods; RibOxi-seq, RiboMeth-seq and 2'-Me-seq. This is the first study using three genome-wide mapping approaches on rRNA from the same species showing the discrepancy among the methods.

The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA.

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction.

Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions.

The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq.

Control of mRNA Splicing by Intragenic RNA Activators of Stress Signaling: Potential Implications for Human Disease.

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100-200 nucleotides in length within primary transcripts of cellular genes, exemplified by the gene and fetal and adult genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA.

Small nucleolar RNAs controlling rRNA processing in Trypanosoma brucei.

In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps.

The Canonical Poly (A) Polymerase PAP1 Polyadenylates Non-Coding RNAs and Is Essential for snoRNA Biogenesis in Trypanosoma brucei.

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown.

PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly.

Short elements in mammalian mRNA can control gene expression by activating the RNA-dependent protein kinase PKR that attenuates translation by phosphorylating cytoplasmic eukaryotic initiation factor 2α (eIF2α). We demonstrate a novel, positive role for PKR activation and eIF2α phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35.

Exosome secretion affects social motility in Trypanosoma brucei.

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes.

A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream.

The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability.

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation.

Dynamic refolding of IFN-gamma mRNA enables it to function as PKR activator and translation template.

Interferon-gamma mRNA activates the RNA-dependent protein kinase PKR, which in turn strongly attenuates translation of interferon-gamma mRNA. Unlike riboswitches restricted to noncoding regions, the interferon-gamma RNA domain that activates PKR comprises the 5' UTR and 26 translated codons. Extensive interferon-gamma coding sequence is thus dedicated to activating PKR and blocking interferon-gamma synthesis.

Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis.

An mRNA structure that controls gene expression by binding FMN.

The RFN element is a highly conserved domain that is found frequently in the 5'-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. We report that this domain serves as the receptor for a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. Our results also indicate that in Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination of the ribDEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA.