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1.  Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog 
Eukaryotic Cell  2014;13(7):896-908.
Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.
doi:10.1128/EC.00071-14
PMCID: PMC4135740  PMID: 24839125
2.  Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab 
Cancer Biology & Therapy  2013;14(6):481-491.
The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (CtxR) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in CtxR clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all CtxR clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of CtxR cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.
doi:10.4161/cbt.24342
PMCID: PMC3813564  PMID: 23760490
AKT; EGFR; MK-2206; cetuximab; acquired cetuximab-resistance; non-small cell lung cancer; MAPK
3.  The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants 
PLoS Genetics  2014;10(2):e1004007.
Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.
Author Summary
Some plant trypanosomes, single-celled organisms living in phloem sap, are responsible for important palm diseases, inducing frequent expensive and toxic insecticide treatments against their insect vectors. Other trypanosomes multiply in latex tubes without detriment to their host. Despite the wide range of behaviors and impacts, these trypanosomes have been rather unceremoniously lumped into a single genus: Phytomonas. A battery of molecular probes has been used for their characterization but no clear phylogeny or classification has been established. We have sequenced the genomes of a pathogenic phloem-specific Phytomonas from a diseased South American coconut palm and a latex-specific isolate collected from an apparently healthy wild euphorb in the south of France. Upon comparison with each other and with human pathogenic trypanosomes, both Phytomonas revealed distinctive compact genomes, consisting essentially of single-copy genes, with the vast majority of genes shared by both isolates irrespective of their effect on the host. A strong cohort of enzymes in the sugar metabolism pathways was consistent with the nutritional environments found in plants. The genetic nuances may reveal the basis for the behavioral differences between these two unique plant parasites, and indicate the direction of our future studies in search of effective treatment of the crop disease parasites.
doi:10.1371/journal.pgen.1004007
PMCID: PMC3916237  PMID: 24516393
4.  Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab 
Oncogene  2010;30(5):561-574.
KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux®) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, sprycel®) a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src Family Kinases. We analyzed 16 CRC lines for: 1) KRAS mutation status, 2) dependence on mutant KRAS signaling, 3) expression level of EGFR and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo, and HCT116) cell lines, and two KRAS wild type cell lines (SW48 and CaCo2). In vitro, using Poly-D-Lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab whereas parental controls showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater anti-proliferative effect on KRAS mutant line as compared to either agent alone both in vitro and in vivo. To investigate potential mechanisms for this anti-proliferative response in the combinatorial therapy we performed Human Phospho-kinase Antibody Array analysis measuring the relative phosphorylation levels of phosphorylation of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the classical MAPK pathway, AKT/mTOR pathway and the family of STAT transcription factors when compared to the untreated control or monotherapy treatments. Next we analyzed tumor growth with cetuximab, dasatinib or the combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an anti-tumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation as measured by Ki67 and higher rates of apoptosis as measured by TUNEL. The data presented herein indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key-signaling pathways. Further, these results suggest that signaling via the EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. This data strengthen the rationale for clinical trials in this genetic setting combining cetuximab and dasatinib.
doi:10.1038/onc.2010.430
PMCID: PMC3025039  PMID: 20956938
Cetuximab; resistance; KRAS mutation; dasatinib; EGFR; SRC; colorectal cancer
5.  Spliced Leader RNAs, Mitochondrial Gene Frameshifts and Multi-Protein Phylogeny Expand Support for the Genus Perkinsus as a Unique Group of Alveolates 
PLoS ONE  2011;6(5):e19933.
The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80–83 nt in P. marinus, and ∼83 nt in P. chesapeaki, significantly larger than the typical ≤56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized ‘marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata.
doi:10.1371/journal.pone.0019933
PMCID: PMC3101222  PMID: 21629701
6.  2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family 
Nucleic Acids Research  2011;39(11):4756-4768.
The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.
doi:10.1093/nar/gkr038
PMCID: PMC3113572  PMID: 21310715
7.  Dinoflagellate Spliced Leader RNA Genes Display a Variety of Sequences and Genomic Arrangements 
Molecular Biology and Evolution  2009;26(8):1757-1771.
Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5′-end of a small noncoding RNA, the SL RNA, to the 5′-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50–60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50–60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA–only, 5S rRNA–only, and mixed SL RNA–5S rRNA (SL–5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL–5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA–only array was also found in three dinoflagellates, along with two SL–5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL–5S cluster genomic organizations are an “ancient” and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.
doi:10.1093/molbev/msp083
PMCID: PMC2734150  PMID: 19387009
Dinoflagellate; SL RNA; complex genomic arrangement
8.  Hypermethylated cap 4 maximizes Trypanosoma brucei translation 
Molecular microbiology  2009;72(5):1100-1110.
Summary
Through trans-splicing of a 39-nt Spliced Leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′-O-ribose methyltransferases, TbMTr1, TbMTr2, and TbMTr3, reveal distinct roles for four 2′-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells, however attempts at double knockouts resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable to wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.
PMCID: PMC2859698  PMID: 19504740
gene knockout; methyltransferase; ribose 2′-O-methylation; SL RNA; spliced leader; trans-splicing
9.  Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex ▿ ‡  
Molecular and Cellular Biology  2008;29(5):1202-1211.
Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.
doi:10.1128/MCB.01496-08
PMCID: PMC2643836  PMID: 19103757
10.  The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei▿ †  
Molecular and Cellular Biology  2007;27(17):6084-6092.
mRNA cap 1 2′-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2′-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2′-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2′-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2′-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3′-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.
doi:10.1128/MCB.00647-07
PMCID: PMC1952150  PMID: 17606627
11.  Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei†  
Eukaryotic Cell  2006;5(6):905-915.
In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m7G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A2 and is not required for subsequent steps; TbMT511 methylates C3, without which U4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m7G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A2, C3, and U4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.
doi:10.1128/EC.00080-06
PMCID: PMC1489268  PMID: 16757738
12.  3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates†  
Molecular and Cellular Biology  2004;24(23):10390-10396.
In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3′ extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3′→5′ exonuclease required for SL RNA 3′-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3′ processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3′→5′ proofreading ɛ subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3′ extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3′ extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3′-end formation is a multistep process in which SNIP provides the ultimate 3′-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.
doi:10.1128/MCB.24.23.10390-10396.2004
PMCID: PMC529039  PMID: 15542846
13.  SmD1 Is Required for Spliced Leader RNA Biogenesis 
Eukaryotic Cell  2004;3(1):241-244.
The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans-splicing competence. In Trypanosoma brucei, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3′-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U28 pseudouridylation was unaffected.
doi:10.1128/EC.3.1.241-244.2004
PMCID: PMC329508  PMID: 14871954
14.  Exportin 1 Mediates Nuclear Export of the Kinetoplastid Spliced Leader RNA 
Eukaryotic Cell  2003;2(2):222-230.
The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3′ extension removal and 5′ methylation of nucleotide U4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.
doi:10.1128/EC.2.2.222-230.2003
PMCID: PMC154853  PMID: 12684371
16.  Representing nested semantic information in a linear string of text using XML. 
XML has been widely adopted as an important data interchange language. The structure of XML enables sharing of data elements with variable degrees of nesting as long as the elements are grouped in a strict tree-like fashion. This requirement potentially restricts the usefulness of XML for marking up written text, which often includes features that do not properly nest within other features. We encountered this problem while marking up medical text with structured semantic information from a Natural Language Processor. Traditional approaches to this problem separate the structured information from the actual text mark up. This paper introduces an alternative solution, which tightly integrates the semantic structure with the text. The resulting XML markup preserves the linearity of the medical texts and can therefore be easily expanded with additional types of information.
PMCID: PMC2244450  PMID: 12463856
18.  Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids 
Molecular and Cellular Biology  1999;19(2):1595-1604.
Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.
PMCID: PMC116087  PMID: 9891092
19.  Three Small Nucleolar RNAs Identified from the Spliced Leader-Associated RNA Locus in Kinetoplastid Protozoans 
Molecular and Cellular Biology  1998;18(8):4409-4417.
First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and ∼450 or ∼350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2′-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.
PMCID: PMC109026  PMID: 9671450
20.  Screening for Novel Antimicrobials from Encoded Combinatorial Libraries by Using a Two-Dimensional Agar Format 
A sensitive lawn-based format has been developed to screen bead-tethered combinatorial chemical libraries for antimicrobial activity. This method has been validated with beads linked to penicillin V via a photocleavable chemical linker in several analyses including a spike-and-recover experiment. The lawn-based screen sensitivity was modified to detect antibacterial compounds of modest potency, and a demonstration experiment with a naive combinatorial library of over 46,000 individual triazines was evaluated for antibacterial activity. Numerous hits were identified, and both active and inactive compounds were resynthesized and confirmed in traditional broth assays. This demonstration experiment suggests that novel antimicrobial compounds can be easily identified from very large combinatorial libraries of small, nonpeptidic compounds.
PMCID: PMC105620  PMID: 9624492

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