Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei.
ABSTRACT: Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.
Project description:At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, 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 TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) 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.
Project description:Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle.Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA.Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.
Project description:In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicer-independent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.
Project description:Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes. A genetic knockout of AGO1 leads to a loss of RNAi, and concomitantly, endogenous retroposon-derived siRNAs as well as siRNAs derived from transgenic double-stranded RNA are reduced to almost undetectable levels. Furthermore, AGO1 deficiency leads to an increase in retroposon transcript abundance via mechanisms operating at the transcriptional level and at the RNA stability level. Our results suggest that AGO1 function is required for production and/or stabilization of siRNAs and provide the first evidence for an Argonaute protein being involved in the regulation of retroposon transcript levels.
Project description:Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L.?braziliensis Argonaute1 (LbrAGO1). In contrast to T.?brucei, Leishmania siRNAs are sensitive to 3' end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2'-O-methyltransferase, which modifies small RNA 3' ends. Consistent with this observation,?~20% of siRNA 3' ends carry non-templated uridines. Thus siRNA biogenesis, and most likely their metabolism, is different in these organisms. Similarly to T.?brucei, putative mobile elements and repeats constitute the major Leishmania siRNA-producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T.?brucei, no siRNAs were detected from other genomic regions with the potential to form double-stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism-specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.
Project description:The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.
Project description:In insects, the RNA interference (RNAi) pathway plays a major role in antiviral responses, as shown against many RNA viruses. The response includes the cleavage of double-stranded RNA genome or intermediates, produced during replication, into viral short interfering RNAs (v-siRNAs). Using deep sequencing, we found that a large number of small reads of ?20 nucleotides from Helicoverpa armigera larvae infected with Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) were mapped to certain open reading frames in the viral genome (hot spots) that are mostly structural and auxiliary late genes. After excluding the possibility of these small RNAs being microRNAs, it was determined that Dicer-2, the main enzyme implicated in the RNAi response in insects, is involved in the generation of v-siRNAs. In Dicer-2- but not Dicer-1-silenced cells, higher transcript levels of the hot spot genes were detected, and as a consequence the virus replicated more efficiently. The results suggest that the viral transcripts are degraded by the RNAi response of the host. This may, however, be to the advantage of the virus by preventing overreplication of the virus, which may otherwise lead to the premature death of the host cells.
Project description:RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.
Project description:The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3'-5' exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.
Project description:RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.