The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal ... more The nucleolus is a sub-nuclear 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. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programme...
Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The la... more Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The large-subunit rRNA of these organisms undergoes unique processing events absent in other eukaryotes. Recently, small nucleolar RNAs (snoRNAs) that mediate these specific cleavages were identified. Trypanosomatid rRNA is rich in RNA modifications such as 2'-O-methylation (Nm) and pseudouridylation (Ψ) that are also guided by these snoRNAs. A subset of these modifications is developmentally regulated and increased in the parasite form that propagates in the mammalian host. Such hypermodification contributes the temperature adaptation and hence infectivity during cycling of the parasite. rRNA processing and modification should be considered promising drug targets for fighting the diseases caused by these parasites.
The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an inse... more 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. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth ...
The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known ... more The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms which are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases 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. In trypanosomes all mRNAs, as well as non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the small nucleolar RNAs and long non-coding RNAs. Under silencing of either PAP1 or PAP2 the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both ...
Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. ... more 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. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonst...
The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animal... more 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. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modi...
The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal ... more The nucleolus is a sub-nuclear 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. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programme...
Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The la... more Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The large-subunit rRNA of these organisms undergoes unique processing events absent in other eukaryotes. Recently, small nucleolar RNAs (snoRNAs) that mediate these specific cleavages were identified. Trypanosomatid rRNA is rich in RNA modifications such as 2'-O-methylation (Nm) and pseudouridylation (Ψ) that are also guided by these snoRNAs. A subset of these modifications is developmentally regulated and increased in the parasite form that propagates in the mammalian host. Such hypermodification contributes the temperature adaptation and hence infectivity during cycling of the parasite. rRNA processing and modification should be considered promising drug targets for fighting the diseases caused by these parasites.
The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an inse... more 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. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth ...
The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known ... more The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms which are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases 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. In trypanosomes all mRNAs, as well as non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the small nucleolar RNAs and long non-coding RNAs. Under silencing of either PAP1 or PAP2 the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both ...
Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. ... more 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. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonst...
The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animal... more 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. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modi...
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Papers by Vaibhav Chikne