Hemozoin inhibiting 2-phenylbenzimidazoles active against malaria parasites.

The 2-phenylbenzimidazole scaffold has recently been discovered to inhibit ß-hematin (synthetic hemozoin) formation by high throughput screening. Here, a library of 325,728 N-4-(1H-benzo[d]imidazol-2-yl)aryl)benzamides was enumerated, and Bayesian statistics used to predict ß-hematin and Plasmodium falciparum growth inhibition. Filtering predicted inactives and compounds with negligible aqueous solubility reduced the library to 35,124. Further narrowing to compounds with terminal aryl ring substituents only, reduced the library to 18, 83% of which were found to inhibit ß-hematin formation <100 µM and 50% parasite growth <2 µM. Four compounds showed nanomolar parasite growth inhibition activities, no cross-resistance in a chloroquine resistant strain and low cytotoxicity. QSAR analysis showed a strong association of parasite growth inhibition with inhibition of ß-hematin formation and the most active compound inhibited hemozoin formation in P. falciparum, with consequent increasing exchangeable heme. Pioneering use of molecular docking for this system demonstrated predictive ability and could rationalize observed structure activity trends.

A simple model system for age-dependent DNA damage and cancer.

Aging is the major risk factor for many human cancers. However, the mechanisms responsible for the effect of aging on tumor incidence are poorly understood, in part because few model systems are available to study age-dependent genomic instability. Furthermore, the role of DNA mutations in "normal aging" and "life span extension" is unclear. Our laboratory has developed a novel method to study aging in yeast based on the survival of non-dividing populations (chronological life span). Two major pathways have been identified that control chronological aging: the Ras/PKA/Msn2/4 and the Sch9 pathways. The downregulation of either of them promotes life span extension. Importantly, similar pathways (insulin/IGF-I-like), regulate longevity in higher eukaryotes suggesting a common evolutionary origin for the life span-regulatory mechanisms. Moreover, both Ras and Sch9 are functional homologs of two major mammalian oncogenes (Ras and Akt), which underlines the close link between cancer and aging. By combining chronological life span with simple assays for the detection of DNA mutations and dedifferentiation we have developed a powerful system to identify genes that regulate genomic instability and understand the fundamental mechanisms that may be responsible for age-dependent DNA mutations and cancer in mammals. Here, we describe the use of this system to monitor the age-dependent accumulation of different types of DNA mutations including base substitutions, frame-shift mutations, and gross chromosomal rearrangements (GCRs).

Susceptibilidade de adultos de Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) a fungicidas utilizados no controle de doenças da macieira / Susceptibility of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) adults to fungicides used to control apple diseases

Este trabalho objetivou avaliar a susceptibilidade em condições de laboratório de adultos do parasitóide de ovos Trichogramma pretiosum Riley a fungicidas utilizados na Produção Integrada da Maçã (PIM). Os bioensaios foram conduzidos utilizando-se protocolos padrões da Organização Internacional para o Controle Biológico (IOBC), Seção Regional Paleártica Oeste (WPRS). Foram testados doze fungicidas nas respectivas doses (g ou ml ingrediente ativo/100 L) captana 1 (0,115), captana 2 (0,120), cresoxim-metílico (0,010), enxofre 1 (AG) (0,480), enxofre 2 (0,480), folpete (0,105), mancozebe (0,160), piraclostrobina (0,010), tebuconazol (0,010), tetraconazol (0,005), tiofanato-metílico (0,050) e triforina (0,024). Adicionalmente, utilizou-se água destilada como testemunha negativa e o inseticida triclorfom (0,150) como testemunha positiva. Adultos do parasitóide foram expostos a resíduos dos tratamentos depositados sobre placas de vidro, sendo avaliadas as reduções no parasitismo em relação à testemunha (somente água). Cada tratamento teve quatro repetições. Os resultados permitiram classificar os fungicidas quanto ao impacto sobre os parasitóides em quatro classes: 1, inócuo (< 30 por cento); 2, levemente nocivo (30-79 por cento); 3, moderadamente nocivo (80-99 por cento); e 4, nocivo (> 99 por cento). Dentre os fungicidas testados, 75 por cento foram considerados seletivos (classes 1 e 2) ao parasitóide. Os fungicidas captana 1, captana 2, cresoxim-metílico, folpete, piraclostrobina, tebuconazol, tiofanato-metílico e triforina foram inócuos; mancozebe foi levemente nocivo; enxofre 1 (AG) e tetraconazol foram moderadamente nocivos e enxofre 2 foi nocivo. Estes resultados devem ser levados em consideração quando utilizamos fungicidas no controle de doenças fúngicas em pomares de macieira, preservando assim, o parasitóide de ovos T. pretiosum.

[Susceptibility of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) adults to fungicides used to control apple diseases]. / Susceptibilidade de adultos de Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) a fungicidas utilizados no controle de doengas da macieira.

This study evaluated the susceptibility under laboratory conditions of Trichogrammapretiosum Riley adults to fungicides recommended by the Integrated Production of Apple (IPA). The bioassays were carried out using the International Organization for Biological Control (IOBC), West Palearctic Regional Section (WPRS) standard protocols. Twelve selected fungicides were studied in the doses (g or ml active ingredient/100 L) captan 1 (0.115), captan 2 (0.120), kresoxim-methyl (0.010), sulphur 1 (AG) (0.480), sulphur 2 (0.480), folpet (0.105), mancozeb (0.160), pyraclostrobin (0.010), tebuconazole (0.010), tetraconazole (0.005), thiophanate-methyl (0.050) and triforine (0.024). Distilled water was used as the blank treatment and the insecticide triclorfon (0.150) as a positive control. The parasitoids were exposed to dry residues applied on glass plates. The reduction in the capacity of parasitism was used to measure the effect of the chemical in comparison to the blank treatment. Each treatment was replicated four times. The results allowed us to classify the fungicides tested in four categories: 1, harmless (< 30%); 2, slightly harmful (30-79%); 3, moderately harmful (80-99%); and 4, harmful (> 99%). 75% of the tested substances were classified as selective (classes 1 and 2) to the parasitoid. The fungicides captan 1, captan 2, kresoxim-methyl, folpet, pyraclostrobin, tebuconazole, thiophanate-methyl and triforine were harmless; mancozeb was slightly harmful; sulphur 1 (AG) and tetraconazole were moderately harmful and sulphur 2 was harmful. These findings should be taken into account when selecting fungicides to spray apple orchards against fungi diseases to preserve the egg parasitoid T. pretiosum.

Biologia comparada de populações de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) em folhas de milho e arroz / Compared biology of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) populations in corn and rice leaves

O objetivo do trabalho foi estudar a biologia de populações de Spodoptera frugiperda (J.E. Smith) em folhas de milho e arroz irrigado. Foram coletadas lagartas de quatro populações no Rio Grande do Sul: em áreas isoladas (distanciadas em mais de 300 km), municípios de Santa Rosa (M/SR) e Uruguaiana (A/U), tradicionalmente produtores de milho e arroz irrigado, respectivamente; e, em áreas adjacentes, município de Pelotas, que produz milho (M/P) e arroz irrigado (A/P) lado a lado. Individualizaram-se 150 lagartas em tubos de vidro contendo folhas do híbrido de milho Pioneer 30F33 e do cultivar de arroz irrigado Pelota, em condições controladas de temperatura (25 ± 1ºC), umidade relativa (70 ± 10 por cento) e fotofase (14h). Por ocasião da emergência dos adultos, 20 casais foram individualizados em gaiolas cilíndricas de PVC revestidas internamente com papel jornal, e alimentados com solução aquosa de mel a 10 por cento. As populações de S. frugiperda originárias do milho e arroz, independente do local de coleta, apresentaram necessidades fisiológicas intrínsecas e que são evidenciadas nos diferentes parâmetros biológicos avaliados. As populações M/SR e M/P (cultura do milho) e A/U e A/P (cultura do arroz irrigado), não apresentaram diferenças fisiológicas. Diante dos resultados, conclui-se que ambos biótipos "milho" e "arroz" de S. frugiperda ocorrem no Rio Grande do Sul.

The objective of this work was to analyze the biology of Spodoptera frugiperda (J.E.Smith) populations in corn and irrigated rice leaves. Caterpillars from four populations from Rio Grande do Sul state were collected from isolated areas (distant more than 300 km apart), from the counties of Santa Rosa (M/SR) and Uruguaiana (A/U), traditional areas of corn and irrigated rice production, respectively, and in adjacent areas in Pelotas county, where corn (M/P) and rice (A/P) aregrown side by side. In the laboratory, 150 caterpillars were individualized in glass vials containing leaves of the Pioneer 30F33 corn hybrid and the cultivar of the irrigated rice Pelota, under controlled temperature (25 ± 1ºC), relative humidity (70 ± 10%) and photophase (14h). After adult emergence, 20 couples were individualized in cylindrical PVC cages, covered inside with paper towels, and fed with a 10% honey aqueous solution. All populations of S. frugiperda collected from corn and rice, regardless their place of origin, showed unique physiological needs, as evidenced by the different biological parameters evaluated. The Populations M/SR and M/P (maize) and A/U and A/P (irrigatedrice), did not differ physiologically. Based on the results, both “corn” and “rice” S. frugiperda biotypes are present in Rio Grande do Sul.

Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae.

Mutations in RAS2, CYR1, and SCH9 extend the chronological life span in Saccharomyces cerevisiae by activating stress-resistance transcription factors and mitochondrial superoxide dismutase (Sod2). Here we show that mutations in CYR1 and SCH9 also extend the replicative life span of individual yeast mother cells. However, the triple deletion of stress-resistance genes MSN2/MSN4 and RIM15, which causes a major decrease in chronological life span, extends replicative life span. Similarly, the overexpression of superoxide dismutases, which extends chronological survival, shortens the replicative life span and prevents budding in 30-40% of virgin mother cells. These results suggest that stress-resistance transcription factors Msn2/Msn4 negatively regulate budding and the replicative life span in part by increasing SOD2 expression. The role of superoxide dismutases and of other stress-resistance proteins in extending the chronological life span of yeast, worms, and flies indicates that the negative effect of Sod2, Msn2/Msn4/Rim15 on the replicative life span of S. cerevisiae is independent of aging.

Consumo e Utilização de Alimento por Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) Originária de Diferentes Regiões do Rio Grande do Sul, das Culturas do Milho e do Arroz Irrigado / Consumption and Utilization of Food by Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) Native to Different Areas in Rio Grande do Sul, from Corn and Irrigated Rice

The goal of this work was to determine by means of consumption and utilization of natural food, the existence of strains of Spodoptera frugiperda (J.E. Smith)in different areas of corn and irrigated rice in Rio Grande do Sul. Four populations were collected: one in Santa Rosa, traditional area of corn cropping; one in Uruguaiana, traditional area of rice; and two in Pelotas, where corn and rice are planted side by side. In the laboratory, 20 larvae were individualized (second generation) and kept in petri dishes, in BOD, at 25°C temperature, 70 ± 10 percent RH and 14h photophase. The larvae were fed on leaves of their respective hosts, hybrid of corn BRS 8330 and the cultivar of irrigated rice Embrapa 6-Chuí. The leaves of both hosts were submitted to bromatological analysis. The dry weight of the larva at maximum development, food consumed and feces eliminated were determined and the nutritional ratios were calculated: relative consumption ratio (RCR), relative metabolic ratio (RMR), relative growth ratio (RGR), approximate digestibilidade (AD), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD) and the metabolic cost (100 - ECD). Rice was more suitable as food for S. frugiperda; it was ingested in smaller amount, presented smaller 100 - ECD and larger ECI and ECD. A strong possibility of existence of two strains of S. frugiperda is evidenced in the State of Rio Grande do Sul, the corn and rice strains, which are morphologically similar but physiologically different.

O objetivo do trabalho foi verificar, através de medidas de consumo e utilização de alimento natural, a existência de raças de Spodoptera frugiperda (J.E. Smith) nas culturas do milho e do arroz irrigado no Rio Grande do Sul. Foram coletadas quatro populações: uma em Santa Rosa, região tradicionalmente produtora de milho; uma em Uruguaiana, região tradicionalmente produtora de arroz; e duas em Pelotas, região que produz milho e arroz lado a lado (uma em milho e outra em arroz). Individualizaram-se 20 lagartas de cada população (segunda geração) em placas de Petri, mantidas em BOD à temperatura de 25°C, UR 70 ± 10 por cento e fotofase de 14h. As lagartas foram alimentadas com folhas de seus respectivos hospedeiros, o híbrido de milho BRS 8330 e a cultivar de arroz irrigado Embrapa 6-Chuí. As folhas de ambos hospedeiros foram submetidas a análise bromatológica. Foram determinados o peso seco da lagarta no máximo desenvolvimento, do alimento consumido, e das fezes eliminadas, e calculados os índices nutricionais: taxa de consumo relativo (RCR), taxa metabólica relativa (RMR), taxa de crescimento relativo (RGR), digestibilidade aproximada (AD), eficiência de conversão do alimento ingerido (ECI), eficiência de conversão do alimento digerido (ECD) e custo metabólico (100 - ECD). O arroz apresentou-se mais adequado para a alimentação dos insetos, pois foi ingerido em menor quantidade, apresentando menor 100 - ECD, e maior ECI e ECD. Evidencia-se forte possibilidade de existirem duas raças de S. frugiperda no Rio Grande do Sul, a "raça do milho" e a "raça do arroz", morfologicamente iguais e fisiologicamente diferentes.

Regulation of longevity and stress resistance: a molecular strategy conserved from yeast to humans?

Recent studies implicate similar proteins in the regulation of longevity in organisms ranging from yeast to mice. Studies in yeast and worms suggest that inactivation of glucose or insulin/insulin-like growth factor-l (IGF-1) signaling pathways extends longevity by causing a shift from a reproductive phase to a non-reproductive maintenance phase involving the expression of many genes. These stress resistance pathways appear to have evolved to induce maintenance systems and promote longevity during periods of starvation. In yeast, mutations that decrease the activity of glucose signaling pathways extend longevity by activating stress resistance transcription factors that regulate the expression of genes involved in antioxidant and heat protection, glycogen storage, protein degradation, DNA repair, and metabolism. A remarkably similar set of proteins regulated by growth factors that control glucose metabolism is implicated in life span extension in worms, and possibly in flies and mice. Studies in worms and flies point to secondary hormones as mediators of the effect of insulin/ IGF-1 signaling on longevity, whereas studies in yeast and mammalian cells indicate that glucose or insulin/ IGF-1 may decrease longevity by directly down-regulating stress resistance genes. In yeast, longevity mutations postpone superoxide toxicity and mitochondrial damage. However, the small life span extension caused by the overexpression of superoxide dismutases and catalase in yeast and flies indicates that increased antioxidant protection alone cannot be responsible for the major life span extension caused by signal transduction mutations. Although we are only beginning to understand the molecular mechanisms that mediate life span extension, the similarities between longevity regulatory pathways in organisms ranging from yeast to mice suggest that insulin/ IGF-1 signaling pathways may also regulate cell damage and longevity in humans.

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.

Regulation of longevity and stress resistance by Sch9 in yeast.

The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans. We screened for long-lived mutants in nondividing yeast Saccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold. Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension. These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly.

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.

Decreasing the dosimetric effects of misalignment when using a mono-isocentric technique for irradiation of head and neck cancer.

PURPOSE: The purpose of this study was to quantify and develop methods to decrease inhomogeneities created with field edge mismatch when using a mono-isocentric beam-split technique. METHODS AND MATERIALS: We validated techniques to determine dose across a half-blocked field edge and quantified potential sources of systematic matchline error. Then, two methods were used to evaluate matchline doses. The first used film dosimetry data from a half-beam field and a spreadsheet. Duplication and reversal provided two columns, each representing a beam-split field edge. Summation simulated perfect abutment and shifting created various gaps and overlaps. The second method involved obtaining dose profiles at midfield along the ray perpendicular to abutted, overlapped, and gapped beam-split fields on six linear accelerators. To enlarge the penumbra, we designed several field edge modifiers, then re-evaluated matchline doses. The field edge modifiers applicability to a 3-field head and neck treatment technique was also examined. RESULTS: Film-determined dose profiles provide similar information across a beam-split field edge as an ionization chamber. With the mono-isocentric beam-split technique, a 4-mm overlap or gap produces inhomogeneities nearly 60% above or below the intended dose. A 2-mm overlap or gap produces inhomogeneities nearly 30% above or below the intended dose. A customized penumbra generator decreased the magnitude of these inhomogeneities to 20% and 10%, respectively. CONCLUSION: The two methods of evaluating matchline dose described above gave similar results. When using the mono-isocentric half-field technique, small misalignments produce worrisome regions of inhomogeneity. Our penumbra generator substantially decreases the magnitude of the dose inhomogeneities, although the volume receiving an inhomogeneous dose increases.

A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP.

The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.

Extremity soft-tissue sarcomas selectively treated with surgery alone.

PURPOSE: This study determined local control (LC), freedom from distant recurrence (FFDR), overall survival (OS), and potential prognostic factors in 34 adult patients with primary extremity or limb girdle soft-tissue sarcoma selectively managed with limb-conservation surgery alone. METHODS AND MATERIALS: The medical records of 34 patients who underwent surgery alone for localized soft-tissue sarcoma of the extremity were reviewed. Median duration of follow-up in survivors was 55 months (range, 24-143). There were 13 (38%) females. Eighteen (53%) of the tumors were low-grade, 15 (44%) were superficial, 15 (44%) were small (5 cm or less), and 16 (47%) involved the distal extremity. RESULTS: Actuarial 5-year LC was 80%, FFDR was 86%, and OS was 82%. All recurrences (local and distant) were in patients with high-grade tumors; their 5-year LC was 60%, FFDR was 71%, and OS was 69%. In 2 patients, metastatic disease developed either concurrent with or after their local recurrence. Univariate analysis revealed better OS, FFDR, and LC for patients with low-grade tumors (p < 0.05). Female patients had significantly better FFDR and OS (p < 0.05). CONCLUSION: It is appropriate to consider withholding irradiation for selected patients with low-grade tumors resected with negative margins if, in the event of a local failure, a function-preserving surgical salvage is anticipated. For patients with high-grade sarcomas, the control of local and distant disease was not acceptable with limb-conservation surgery alone.

Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15 kD protein.

The U5 small ribonucleoprotein particle (snRNP) contains various proteins involved in catalytic activities mediating conformational rearrangements of the spliceosome. We have isolated and characterized the evolutionarily highly conserved human U5 snRNP-specific protein U5-15kD. The crystal structure of U5-15kD determined at 1.4 A resolution revealed a thioredoxin-like fold and represents the first structure of a U5 snRNP-specific protein known so far. With respect to human thioredoxin the U5-15kD protein contains 37 additional residues causing structural changes which most likely form putative binding sites for other spliceosomal proteins or RNA. Moreover, a novel intramolecular disulfide bond replaces the canonical one found in the thioredoxin family. Even though U5-15kD appears to lack protein disulfide isomerase activity, it is strictly required for pre-mRNA splicing in vivo as we demonstrate by genetic depletion of its ortholog in Saccharomyces cerevisiae. Our data suggest that the previously reported involvement of its Schizosaccharomyces pombe ortholog Dim1p in cell cycle regulation is a consequence of its essential role in pre-mRNA splicing.

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.

Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection.

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.