Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA.

Capping of the pre-mRNA 5' end by addition a monomethylated guanosine cap (m(7)G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m(7)G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m(7)G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes.

esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse.

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.

Reduction of PTP1B by RNAi upregulates the activity of insulin controlled fatty acid synthase promoter.

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of IR autophosphorylation and thus has been considered as a major therapeutic target for the treatment of type II diabetes. We use RNA interference technique to downregulate PTP1B expression in hepatoma cell line. A secretory HBV s-antigen was introduced as reporter and driven by mouse fatty acid synthase promoter, which is positively controlled by insulin signaling. Liver-targeted hydrodynamic injection in tail vein was introduced to transfer siRNA (or siRNA expression vector) and reporter plasmid into mouse liver. On fasted/refed and glucose stimulation condition, the HBV s-antigen in sera in RNAi group was higher than that in the negative group. Our results provided evidence that upregulation of insulin signaling by reducing PTP1B liver with RNAi can be a potent diabetes treatment method.

Requirement of the N-terminus for dimer formation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate synthase AroG of Escherichia coli.

The first regulatory step in the synthesis of aromatic amino acids is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). In Escherichia coli, the allosteric DAHPS exists as three isozymes, AroG, AroF and AroH, each independently feedback-inhibited by corresponding end product amino acids, phenylalanine, tyrosine and typtophan. Structural biological evidences have suggested that the N-terminus of AroG is involved in the formation of a putative inhibitor-binding site and feedback inhibition signal transmission. Our previous work showed that a single amino acid residue replacement Ile10Ala or deletion of 15 N-terminal amino acids could lead to a dramatic loss of AroG enzymatic activity (Hu et al. 2003). Here we demonstrate that the deletion of N-terminus prevents the enzyme from forming a dimeric structure, indicating that the N-terminus of AroG plays a critical role in the formation of the essential tight dimeric structure.

Role of a 300-kilodalton nuclear complex in the maturation of Trypanosoma brucei initiator methionyl-tRNA.

tRNAs are transcribed as precursors containing 5' leader and 3' extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA(Met) in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5' leader of initiator tRNA(Met) precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA(Met) containing 5' extensions but processed 3' ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA(Met) molecules with 5' and 3' extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA(Met). Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA(Met), 3' trimming precedes 5' processing.

A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei.

Sequence information on the Trypanosoma brucei genome is rapidly accumulating. As a consequence, there is a need for techniques to analyze gene function systematically. Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epitope-tagged fusion proteins. The approach is based on methodologies developed for Saccharomyces cerevisiae and involves PCR amplification of a reporter cassette using primers containing flanking sequences specific to the target gene. The PCR product is then transfected directly into procyclic T. brucei cells, and homologous recombinants that carry the deleted or tagged target gene are identified.

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. The method of choice today to examine gene function in these organisms is RNA interference (RNAi). Messenger RNA (mRNA) degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. This chapter describes some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA, and analysis of dsRNA and target mRNA.

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Characterization of bacteriophage T3 DNA ligase.

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.

An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei.

Genetic interference by double-stranded RNA (dsRNA) or RNA interference (RNAi) triggers the sequence-specific degradation of cellular transcripts. The mediators of mRNA degradation are small interfering RNAs (siRNAs). Here, we report that in Trypanosoma brucei, 10%-20% of siRNAs cosediment with polyribosomes. Preventing the assembly of ribosomes on mRNAs results in a concomitant decrease of siRNAs sedimenting as high-molecular-weight complexes. We further provide evidence that siRNAs are associated with translating ribosomes and that this association is mediated by a 70-kD ribonucleoprotein complex.