A manganese-dependent ribozyme in the 3′-untranslated region of Xenopus Vg1 mRNA

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3′-UTR of Vg1 and β-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30°C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3′-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3′-end processing of eukaryotic mRNAs

The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution

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

Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery

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

Symplekin and multiple other polyadenylation factors participate in 3′-end maturation of histone mRNAs

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3′ ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation

Two cold shock domain containing proteins trigger the development of infectious Trypanosoma brucei.

Cold shock proteins are members of a family of DNA- and RNA-binding proteins with one or more evolutionarily conserved cold shock domain (CSD). These proteins have a wide variety of biological functions, including DNA-damage repair, mRNA stability, and regulation of transcription, splicing and translation. We previously identified two CSD containing proteins, CSD1 and CSD2, in the protozoan parasite Trypanosoma brucei to be required for RBP6-driven metacyclic production, albeit at different steps of the developmental program. During metacyclogenesis T. brucei undergoes major morphological and metabolic changes that culminate in the establishment of quiescent metacyclic parasites and the acquisition of mammalian infectivity. To investigate the specific role of CSD1 and CSD2 in this process, we ectopically expressed CSD1 or CSD2 in non-infectious procyclic parasites and discovered that each protein is sufficient to produce infectious metacyclic parasites in 24 hours. Domain truncation assays determined that the N-terminal domain, but not the C-terminal domain, of CSD1 and CSD2 was required for metacyclic development. Furthermore, conserved amino acid residues in the CSD of CSD1 and CSD2, known to be important for binding nucleic acids, were found to be necessary for metacyclic production. Using single-end enhanced crosslinking and immunoprecipitation (seCLIP) we identified the specific binding motif of CSD1 and CSD2 as "ANACAU" and the bound mRNAs were enriched for biological processes, including lipid metabolism, microtubule-based movement and nucleocytoplasmic transport that are likely involved in the transition to bloodstream form-like cells

Conserved motifs in both CPSF73 and CPSF100 are required to assemble the active endonuclease for histone mRNA 3′-end maturation

In eukaryotes, the process of messenger RNA 3′-end formation involves endonucleolytic cleavage of the transcript followed by synthesis of the poly(A) tail. The complex machinery involved in this maturation process contains two proteins of the metallo-β-lactamase (MBL) superfamily, the 73 and 100 kDa subunits of the cleavage and polyadenylation specificity factor (CPSF). By using an in vitro system to assess point mutations in these two mammalian proteins, we found that conserved residues from the MBL motifs of both polypeptides are required for assembly of the endonuclease activity that cleaves histone pre-mRNAs. This indicates that CPSF73 and CPSF100 act together in the process of maturation of eukaryotic pre-messenger RNAs, similar to other members of the MBL family, RNases Z and J, which function as homodimers

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

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

Recombinant <i>Tb</i>RIF4 3′-5′ exonuclease activity <i>in vitro</i>.

<p>(<i>A</i>) Titration of GST-RIF4 activity. 1 pmol of a 25 nt-long, 5′-end labelled synthetic dsRNA was incubated with 0.01–30 pmol GST-RIF4 (lanes 4–11; molar ratios from 10∶1 to 1∶30) or 30 pmol GST-<i>Tb</i>RIF4 carrying mutation H472A (lane 12). Lane 3, input. AH, alkaline hydrolysate ladder. M, marker oligonucleotides. Samples were separated on a denaturing 20% polyacrylamide gel. (<i>B</i>) Action of GST-<i>Tb</i>RIF4 on a RNA substrate blocked at the 3′ end of one strand. 1 pmol of a 25 nt-long, 5′-end labelled synthetic dsRNA with a dideoxycytosine residue at the 3′ end of the labelled strand was incubated with 0.03–30 pmol GST-<i>Tb</i>RIF4 (lanes 3–6), or buffer alone (lanes 1 and 2). Samples were separated on a non-denaturing 16% polyacrylamide gel. As a control for the separation of double-stranded and single-stranded species, the substrate incubated with buffer alone was resolved on the same gel without (lane 1, native) or with (lane 2, denat.) heating to 100°C for 2 minutes prior to electrophoresis.</p

siRNAs are not associated with <i>Tb</i>AGO1 in <i>rif4^−/−</i> cells.

<p>(<i>A</i>) Cytoplasmic extracts from cells expressing TAP-tagged <i>Tb</i>AGO1 in a wild-type (lanes 1–4) or <i>rif4^−/−</i> (lanes 5–8) background were immunoprecipitated with IgG beads, and the indicated amounts of supernatant (S, lanes 1 and 5) and immunoprecipitated material (P, lanes 2–4 and 6–8) were analyzed by Western blotting with a polyclonal anti-<i>Tb</i>AGO1 antibody (upper panel, <i>Tb</i>AGO1). CIR147 siRNAs in the soluble and precipitated materials were revealed by Northern hybridization (middle panel, CIR147 siRNA). RNA loading was quantified by hybridization to 5S rRNA (bottom panel, 5S RNA). (<i>B</i>) <i>Tb</i>RIF4 and <i>Tb</i>AGO1 steady-state levels in various cell lines. Total protein extracts from 5×10⁶ cells from various cell lines, as indicated above each lane, were resolved by SDS-PAGE and Western blotted with a polyclonal anti-<i>Tb</i>RIF4 antibody (top panel, <i>Tb</i>RIF4) or a polyclonal anti-<i>Tb</i>AGO1 antibody (middle panel, <i>Tb</i>AGO1). Load; a cross-reacting band to the <i>Tb</i>AGO1 antibody (bottom panel).</p

Analysis of <i>Tb</i>RIF4- and <i>Tb</i>RIF5-mutant cell lines.

<p>(<i>A</i>) Response of <i>rif4^−/−</i> cells and cells expressing <i>Tb</i>RIF4 catalytic mutants or <i>Hs</i>AGO2 to transfection of α-tubulin dsRNA. Cells were electroporated with different amounts (in micrograms) of α-tubulin dsRNA, as indicated above each lane or with 5 µg poly(I-C) (first lane in each panel set), and total RNA was prepared 2 h after electroporation and analyzed by Northern hybridization with an α-tubulin-specific probe (α-tubulin panels). α-tubulin mRNA hybridization was quantitated by PhosphorImager analysis, normalized to the load control and expressed as % mRNA remaining, setting as 100% the amount of α-tubulin mRNA present in the samples that received poly(I-C). Load; hybridization to fumarate hydratase mRNA. (<i>B</i>) Steady-state levels of repeat-derived (CIR147) transcripts are increased in <i>rif4^−/−</i> cells but not <i>rif5^−/−</i> cells. Total RNA isolated from various cell lines, as indicated above each lane, was analyzed by Northern hybridization with a CIR147-specific probe. Load; hybridization to α-tubulin mRNA. Filled squares indicate the positions of the large ribosomal RNAs, as determined by methylene blue staining of the membranes. (<i>C</i>) Semi-quantitative RT-PCR analysis of CIR147 transcript levels. cDNA derived from various cell lines (as indicated above each lane; lanes 1–10) was used as a template for 22 cycles of PCR using oligonucleotides that amplify the CIR147 tandem repeat transcripts, producing a ladder of fragments (top two panels), or a portion of histone H4 (bottom two panels). Dilutions of genomic DNA were used as a positive control and an indicator for non-saturated PCR (lanes 11–13); mock cDNA synthesis without reverse transcriptase (second and fourth panels, -RT) served as a negative control. <i>(D)</i> Cells were visualized using differential interference microscopy (DIC), GFP imaging, and Hoechst (Nucleus and Kinetoplast are indicated by N and K, respectively); GFP and Hoechst images were merged (merge).</p