Biomedical subjects

G Dreyfuss

Publications and source records attributed to G Dreyfuss.

At least 55 records · Page 3Linked to original sources

PubMed2001

Characterization of functional domains of the SMN protein in vivo.

The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.

Amino Acid Sequence↗

PubMed2001

Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex.

Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm.

3' Untranslated Regions↗

PubMed2001

Nuclear mRNA binding proteins couple pre-mRNA splicing and post-splicing events.

New information about the pathway of eukaryotic gene expression indicates that many of the steps in this pathway are functionally interconnected. An important link has recently emerged between pre-mRNA splicing and the post-splicing events such as mRNA export and mRNA decay. Recent results reveal that the coupling is mediated by a novel group of nuclear mRNA-binding proteins that are recruited to the mRNAs by spiceosome. These proteins, including Y14, Aly/REF, RNPS1, SRm160, and DEK, are assembled into a stable complex near exon-exon junctions of spliced mRNAs. Several of them persist in their attachment to mRNAs in the cytoplasm thus communicating the history of splicing to the downstream events. The detailed mechanism of coupling and the factors that mediate these processes remain to be determined in the coming years.

Exons↗

PubMed2001

The survival of motor neurons (SMN) protein interacts with the snoRNP proteins fibrillarin and GAR1.

BACKGROUND: The survival of motor neurons (SMN) protein is the protein product of the spinal muscular atrophy (SMA) disease gene. SMN and its associated proteins Gemin2, Gemin3, and Gemin4 form a large complex that plays a role in snRNP assembly, pre-mRNA splicing, and transcription. The functions of SMN in these processes are mediated by a direct interaction of SMN with components of these machineries, such as Sm proteins and RNA helicase A. RESULTS: We show that SMN binds directly to fibrillarin and GAR1. Fibrillarin and GAR1 are specific markers of the two classes of small nucleolar ribonucleoprotein particles (snoRNPs) that are involved in posttranscriptional processing and modification of ribosomal RNA. SMN interaction requires the arginine- and glycine-rich domains of both fibrillarin and GAR1 and is defective in SMN mutants found in some SMA patients. Coimmunoprecipitations demonstrate that the SMN complex associates with fibrillarin and with GAR1 in vivo. The inhibition of RNA polymerase I transcription causes a transient redistribution of SMN to the nucleolar periphery and loss of fibrillarin and GAR1 colocalization with SMN in gems. Furthermore, the expression of a dominant-negative mutant of SMN (SMNDeltaN27) causes snoRNPs to accumulate outside of the nucleolus in structures that also contain components of gems and coiled (Cajal) bodies. CONCLUSIONS: These findings identify fibrillarin and GAR1 as novel interactors of SMN and suggest a function for the SMN complex in the assembly and metabolism of snoRNPs. We propose that the SMN complex performs functions necessary for the biogenesis and function of diverse ribonucleoprotein complexes.

Arginine↗

PubMed2001

The Y14 protein communicates to the cytoplasm the position of exon-exon junctions.

We recently described an RNA-binding protein, Y14, that binds preferentially to spliced mRNAs and persists in the cytoplasm. Y14 is part of a multi-protein complex that also contains the mRNA export factor TAP. This suggests that splicing imprints the mRNA with a unique set of proteins that communicate the history of the transcript to the cytoplasm. Here, using microinjection of pre-mRNAs into Xenopus oocyte nuclei followed by immunoprecipitation of RNase-fragmented mRNAs from the cytoplasm, we show that Y14 is stably bound to sequences immediately upstream of exon-exon junctions. This feature appears to be unique to Y14. Using monoclonal antibodies that we produced against Aly/REF, another component recently reported to be an mRNA export factor, we show that Aly/REF is associated with spliced mRNAs in the nucleus but is not detectable on mRNAs in the cytoplasm. Thus, we propose that the splicing- dependent binding of Y14 provides a position-specific molecular memory that communicates to the cytoplasm the location of exon and intron boundaries. This novel mechanism may play an important role in post-splicing events.

Active Transport, Cell Nucleus↗

PubMed2001

A functional interaction between the survival motor neuron complex and RNA polymerase II.

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex-associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNDeltaN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

Animals↗

PubMed2001

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.

Animals↗

PubMed2001

The definitive and intermediate hosts of Fasciola hepatica in the natural watercress beds in central France.

Field investigations were carried out over a two-year period in 52 natural watercress beds located in the Limousin region of central France to list the mammal and bird species that frequented these sites. This enabled detection of the definitive hosts of Fasciola hepatica and determination of the prevalence of natural infection in snails. A total of 13 mammal and five bird species were listed in these watercress beds. Adult flukes were found in Lepus capensis (39.2%), Oryctolagus cuniculus (42.0%), and Sylvilagus floridanus (25.0%). No infection with F. hepatica was noted in the five species of rodents studied. Snails infected with F. hepatica were found in 14 watercress beds. The global prevalence of natural infection was 1.1% in Lymnaea truncatula and 0.3% in L. glabra. Among the other trematode larval forms detected, the most frequent was Haplometra cylindracea (0.5%). In the Limousin region, the presence of hares and rabbits in watercress beds ensured the continuation of the F. hepatica life cycle and permitted the subsequent infection of humans when this wild watercress was eaten.

Animals↗

PubMed2001

Fasciola hepatica: the characteristics of experimental infections in Lymnaea truncatula subjected to miracidia differing in their mammalian origin.

Experimental infections of Lymaea truncatula, using two susceptible snail populations (Berneuil, or Migné, central France) and four isolates of Fasciola hepatica miracidia differing in their mammalian host of origin (cattle, nutrias, rabbits, or sheep), were performed under laboratory conditions to determine whether the host of origin had an effect on the daily production of cercariae. Snails were each subjected to bimiracidial exposures and were then reared under semi-natural conditions (a constant temperature of 20 degrees C and natural photoperiod). Significantly lower values were noted in the rabbit groups for survival rates at day 30 post-exposure, as well as for prevalences of infection, snail growth. duration of shedding period, and the total numbers of cercariae these snails shed. The total number of cercariae shed by both nutria groups was significantly higher than those recorded in the six other infected groups. In the cattle, rabbit, and sheep (Berneuil only) groups, the peaks in the daily distribution of cercariae occurred between day 2 and day 4 after the first shedding, and the number of cercaria-shedding snails decreased with increasing number of shedding waves. In contrast, in the three other groups, the peaks were only observed between days 20 and 45. Snails shedding their cercariae during nine or more waves were numerous in these last groups. No infradian-type rhythm in the daily distribution of cercarial numbers over the shedding period was noted for any snail group. The highest production of F. hepatica cercariae in both nutria groups would be a consequence of a higher success rate of miracidia when they infected an allopatric population of snails. The absence of an infradian-type rhythm in the distribution of daily cercarial numbers in the eight groups suggests that this rhythm, if it occurs, would only be influenced by temperature and thus be limited to periods with optimal conditions for cercarial shedding.

Animals↗

PubMed2001

More efficient allopatric combinations of Fasciola hepatica and Lymnaea truncatula due to modification of redial development?

Experimental infections of two susceptible French populations of Lymnaea truncatula (Courcelles and Saint Ours) with an allopatric (Fès, Morocco) or a semi-sympatric (Limoges, France) isolate of Fasciola hepatica miracidia were performed to determine the effect of allopatric miracidia on redial and cercarial production. In both populations, cercarial release was significantly greater in allopatric than in semi-sympatric snails. Compared to semi-sympatric snails, the examination of allopatric snails killed from day 14 to day 35 post-exposure demonstrated (1) a significant decrease after day 28 in the number of daughter rediae (R2a group) exiting from the first-appearing mother redia (R1a redia), and (2) the differentiation of numerous daughter rediae (R2b group) in the body of second-appearing mother rediae (R1b group). These experiments demonstrated that the exposure of L. truncatula to an allopatric isolate of miracidia disturbed the usual developmental pattern of redial generations and caused the formation of numerous R2b rediae. The authors hypothesized that the increase in cercarial release noted in allopatric snails would be assured by the rediae from the R2b group after their emergence from the body of R1b mother rediae.

Animals↗

PubMed2001

SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets.

The survival of motor neurons protein (SMN), the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, functions as an assembly factor for snRNPs and likely other RNPs. SMN binds the arginine- and glycine-rich (RG) domains of the snRNP proteins SmD1 and SmD3. Specific arginines in these domains are modified to dimethylarginines, a common modification of unknown function. We show that SMN binds preferentially to the dimethylarginine-modified RG domains of SmD1 and SmD3. The binding of other SMN-interacting proteins is also strongly enhanced by methylation. Thus, methylation of arginines is a novel mechanism to promote specific protein-protein interactions and appears to be key to generating high-affinity SMN substrates. It is reasonable to expect that protein hypomethylation may contribute to the severity of SMA.

Amino Acid Sequence↗

PubMed2001

Metacercarial aggregation in Digenea (Fasciola hepatica and Paramphistomum daubneyi): environmental or species determinism?

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.

Animals↗

PubMed2001

The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins.

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

Arginine↗

PubMed2001

Antitrypanosomal properties of cis-platinum-pentamidine bromide, thiocyanate and seleniocyanate on Trypanosoma brucei brucei mouse and sheep models.

Three organometallic complexes derived from pentamidine were evaluated for their trypanocidal effect on in vivo Trypanosoma brucei brucei models in comparison to pentamidine isethionate as reference compound. On the T. b.brucei mouse model, the most active compound was cis-platinum-pentamidine bromide. This compound was active when subcutaneously administered at the single dose of 1.5 micromol/kg and its chemotherapeutic index was 200 whereas pentamidine isethionate was active at 6 micromol/kg with a chemotherapeutic index of 13, when administered in the same conditions. Cis-platinum-pentamidine bromide was active at 1 mg/kg (1.44 mmoles/kg), in a single dose by subcutaneous route against the early stage of the T. b.brucei Antat 1-9 sheep model. Platinum kinetics in serum showed a Cmax of 0.2 mg/l reached 80 h after the treatment at this dose. Cis-platinum-pentamidine bromide, cis-platinum-pentamidine seleniocyanate, and cis-platinum-pentamidine thiocyanate were distributed in the deep compartment according to a monocompartmental model. In all cases, platinum was eliminated from the serum 700 hours post-treatment. All data obtained from these models show activity on the early stage of the disease and justify further investigations on the late stage of the disease.

Animals↗

PubMed2000

A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN.

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.

Amino Acid Sequence↗

PubMed2000

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1.

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Amino Acid Sequence↗

PubMed2000

Immunohistochemical study of the hnRNP A2 and B1 in the rat forebrain.

Immunohistochemical techniques were employed to examine the distribution of RNA-binding proteins A2 and B1 in the rat forebrain. Intense A2 and B1 immunolabeling were observed in the nucleoplasm of the neurons in the cerebral cortices, hippocampal formation, olfactory regions, caudate-putamen as well as the supraoptic nucleus of hypothalamus. In contrast, within the bed nucleus of the stria terminalis, as well as the medial and lateral habenular nucleus of thalamus, immunoreactivity for both proteins was weak. Within the globus pallidus and thalamic nucleus immunoreactivity for A2 was hardly detectable despite of intense B1 immunolabeling, while within the endopiriform nucleus and lateral and basolateral nucleus of amygdala intensity of B1 immunolabeling was relatively weak compared to A2. Our study suggests that the distribution of A2 and B1 are not constant throughout the forebrain and this diversity may reflect the post-transcriptional regulation of cell-specific gene expression of neuronal cells.

Animals↗

PubMed2000

Specific sequences of the Sm and Sm-like (Lsm) proteins mediate their interaction with the spinal muscular atrophy disease gene product (SMN).

The spinal muscular atrophy disease gene product (SMN) is crucial for small nuclear ribonuclear protein (snRNP) biogenesis in the cytoplasm and plays a role in pre-mRNA splicing in the nucleus. SMN oligomers interact avidly with the snRNP core proteins SmB, -D1, and -D3. We have delineated the specific sequences in the Sm proteins that mediate their interaction with SMN. We show that unique carboxyl-terminal arginine- and glycine-rich domains comprising the last 29 amino acids of SmD1 and the last 32 amino acids of SmD3 are necessary and sufficient for SMN binding. Interestingly, SMN also interacts with at least two of the U6-associated Sm-like (Lsm) proteins, Lsm4 and Lsm6. Furthermore, the carboxyl-terminal arginine- and glycine-rich domain of Lsm4 directly interacts with SMN. This suggests that SMN also functions in the assembly of the U6 snRNP in the nucleus and in the assembly of other Lsm-containing complexes. These findings demonstrate that arginine- and glycine-rich domains are necessary and sufficient for SMN interaction, and they expand further the range of targets of the SMN protein.

Amino Acid Sequence↗