scholarly journals Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis

2008 ◽  
Vol 8 (1) ◽  
pp. 56-68 ◽  
Author(s):  
Avraham Hury ◽  
Hanoch Goldshmidt ◽  
Itai Dov Tkacz ◽  
Shulamit Michaeli
Keyword(s):  
Core Protein ◽  
Rnase H ◽  
Differential Sensitivity ◽  
Minor Effect ◽  
Sm Proteins ◽  
Spliced Leader ◽  
Rna Biogenesis ◽  
Leptomonas Collosoma

ABSTRACT Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position −12 (relative to the 5′ splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

scholarly journals Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

2005 ◽  
Vol 4 (6) ◽  
pp. 1057-1065 ◽  
Author(s):  
M. Wilhelm ◽  
F.-X. Wilhelm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Specific Activity ◽  
Rnase H ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Acidic Domain ◽  
Basic Region

ABSTRACT Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

scholarly journals Xenopus LSm Proteins Bind U8 snoRNA via an Internal Evolutionarily Conserved Octamer Sequence

2002 ◽  
Vol 22 (12) ◽  
pp. 4101-4112 ◽  
Author(s):  
Nenad Tomasevic ◽  
Brenda A. Peculis
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
High Specificity ◽  
Sm Proteins ◽  
U6 Snrna ◽  
Evolutionarily Conserved

ABSTRACT U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2421-2430 ◽  
Author(s):  
A. Walz ◽  
S. McFarlane ◽  
Y.G. Brickman ◽  
V. Nurcombe ◽  
P.F. Bartlett ◽  
...  
Keyword(s):  
Visual System ◽  
Core Protein ◽  
Optic Tract ◽  
Axon Growth ◽  
Axon Elongation ◽  
Axon Extension ◽  
Heparan Sulfates

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.

Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo

2001 ◽  
Vol 358 (3) ◽  
pp. 615-626 ◽  
Author(s):  
John D. SANDY ◽  
Christie VERSCHAREN
Keyword(s):  
Synovial Fluid ◽  
Core Protein ◽  
Globular Domain ◽  
Human Cartilage ◽  
Human Knee ◽  
Synovial Fluids ◽  
Human Joints ◽  
Non Destructive

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE373 and G1-VDIPEN341) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu373–Ala374 bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

Host defense role of platelets: engulfment of HIV andStaphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4021-4029 ◽  
Author(s):  
Tayebeh Youssefian ◽  
Arnaud Drouin ◽  
Jean-Marc Massé ◽  
Josette Guichard ◽  
Elisabeth M. Cramer
Keyword(s):  
Platelet Activation ◽  
Core Protein ◽  
Morphological Evidence ◽  
Subcellular Compartment ◽  
Virus Particles ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Granule Release

Platelets can bind and phagocytose infectious microorganisms and so enable their transport for a prolonged time. To investigate the subcellular events of these interactions, platelets were incubated either with Staphylococcus aureus or with HIV and analyzed by electron microscopy (EM) and immuno-EM. HIV and bacteria internalization occurred exclusively within platelets showing morphological evidence of activation. Platelet activation enhanced the degree of bacterial internalization. Immunolabeling revealed that the engulfing vacuoles and the open canalicular system (OCS) were composed of distinct antigens. The engulfing vacuoles eventually became the site of prominent α-granule release. In platelets incubated with HIV, characteristic endocytic vacuoles were identified close to the plasma membrane, tightly surrounding 1 or 2 HIV particles. Virus particles were also located within the OCS. Immunogold labeling for the viral core protein p24 confirmed the presence of HIV within platelets. Finally, examination of platelets from a patient with acquired immunodeficiency syndrome and high viremia suggested that HIV endocytosis may also occur in vivo.

scholarly journals Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

2014 ◽  
Vol 88 (14) ◽  
pp. 7998-8015 ◽  
Author(s):  
Pak-Guan Teoh ◽  
Zhi-Shun Huang ◽  
Wen-Li Pong ◽  
Po-Chiang Chen ◽  
Huey-Nan Wu
Keyword(s):  
Dengue Virus ◽  
Core Protein ◽  
Virus Production ◽  
Hydrophobic Surface ◽  
Pair Interaction ◽  
Virus Core ◽  
Virion Production

ABSTRACTThe virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal α2 and α4 helices of each monomer. Pairing of α2-α2′ creates a continuous hydrophobic surface, while the α4-α4′ helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the α4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. Thein vitrocharacterization assays further highlighted that the α4-α4′ helix pair conformation is critical in preserving the overall α-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improvesin vitronucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formationin vivo. This study provides the first direct link between the α4-α4′ helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production.IMPORTANCEStructure-based mutagenesis study of the dengue virus core protein (CP) reveals that the α4-α4′ helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected.In vitroinefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation forin vivovirus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by α4-α4′ helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the α4-α4′ helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production.

scholarly journals Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

2010 ◽  
Vol 9 (3) ◽  
pp. 379-386 ◽  
Author(s):  
Nicolas Jaé ◽  
Pingping Wang ◽  
Tianpeng Gu ◽  
Martin Hühn ◽  
Zsofia Palfi ◽  
...  
Keyword(s):  
Essential Role ◽  
Core Protein ◽  
Affinity Purification ◽  
Rnase H ◽  
Essential Step ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

scholarly journals Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

Concomitant neoplasms in the skin and stomach unveil the role of type IV collagen and E-cadherin in mucin core protein 5AC expression in vivo

2015 ◽  
Vol 174 (2) ◽  
pp. 395-397
Author(s):  
H. Hata ◽  
K. Natsuga ◽  
S. Kitamura ◽  
K. Imafuku ◽  
Y. Yamaguchi ◽  
...  
Keyword(s):  
Type Iv Collagen ◽  
Core Protein ◽  
Type Iv ◽  
Mucin Core Protein ◽  
E Cadherin
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