Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.

Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc.

The small nuclear RNA-activating protein complex SNAP(c) is required for transcription of small nuclear RNA genes and binds to a proximal sequence element in their promoters. SNAP(c) contains five types of subunits stably associated with each other. Here we show that one of these polypeptides, SNAP45, also known as PTF delta, localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 lead to a G(2)/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAP(c) subunit, leads to an accumulation of cells with a G(0)/G(1) DNA content. These results are consistent with the proposal that SNAP45 plays two roles in the cell, one as a subunit of the transcription factor SNAP(c) and another as a factor required for proper mitotic progression.

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation.

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly modify in the three groups, novel structural components were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m3G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hi-bernating dormice, were present in much higher amounts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Presence of autoantibodies against small nuclear ribonucleoprotein epitopes in Chagas' patients' sera

We investigated the possibility that Chagas' patients develop an autoimmune response to human UsnRNPs (small nuclear ribonucleoprotein) or Sm epitopes. Using purified human UsnRNPs, we detected anti-human UsnRNPs antibodies in sera from patients suffering from Chagas' disease. The antibodies it-ere also detected using peptide enzyme-linked immunosorbent assays containing the Sm-motif 1 domain. The latter technique showed that 61% (31/51) of the Chagas' patients' sera contained antibodies against Sm-motif I. The detection of anti-UsnRNPs autoantibodies in Chagas patients' sera strongly encourages further studies using animal models to determine how these autoantibodies appear.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m(3)G antibodies

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m(3)G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m(3)G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.