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E Ullu

Publications and source records attributed to E Ullu.

At least 37 records · Page 2Linked to original sources
PubMed1995

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.

Aldehydes↗
PubMed1994

A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.

In trypanosomes, the generation of monocistronic mRNAs from polycistronic precursors is achieved via RNA processing, namely trans-splicing of the spliced leader sequence at the 5' end and cleavage/polyadenylation at the 3' end of the mRNA coding region. Recent evidence raised the intriguing possibility that these two reactions are coupled. To begin a dissection of the signals required for mRNA 5'-end and 3'-end formation and to uncover potential interactions between trans-splicing and polyadenylation, we mutagenized the intergenic region between the beta- and alpha-tubulin genes of Trypanosoma brucei. Block substitutions identified the pyrimidine-rich sequences at the alpha-tubulin 3'-splice-acceptor site as a major determinant for accurate trans-splicing downstream and 3'-end formation upstream. In addition to the utilization of cryptic 3'-splice sites, obliteration of the polypyrimidine tracts led to aberrant poly(A)+ site choice, even in the presence of the wild-type poly(A)+ site and neighboring sequences. Taken together, these results indicate that the polypyrimidine-rich sequences act as a bifunctional element that affects RNA processing both upstream and downstream from itself. This is consistent with the possibility that the polypyrimidine tract is recognized by both the trans-splicing and polyadenylation machineries, either sequentially or simultaneously.

Animals↗
PubMed1994

Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes.

An interesting feature of trypanosome genome organization involves genes transcribed by RNA polymerase III. The U6 small nuclear RNA (snRNA), U-snRNA B (the U3 snRNA homolog), and 7SL RNA genes are closely linked with different, divergently oriented tRNA genes. To test the hypothesis that this association is of functional significance, we generated deletion and block substitution mutants of all three small RNA genes and monitored their effects by transient expression in cultured insect-form cells of Trypanosoma brucei. In each case, two extragenic regulatory elements were mapped to the A and B boxes of the respective companion tRNA gene. In addition, the tRNA(Thr) gene, which is upstream of the U6 snRNA gene, was shown by two different tests to be expressed in T. brucei cells, thus confirming its identity as a gene. This association between tRNA and small RNA genes appears to be a general phenomenon in the family Trypanosomatidae, since it is also observed at the U6 snRNA loci in Leishmania pifanoi and Crithidia fasciculata and at the 7SL RNA locus in L. pifanoi. We propose that the A- and B-box elements of small RNA-associated tRNA genes serve a dual role as intragenic promoter elements for the respective tRNA genes and as extragenic regulatory elements for the linked small RNA genes. The possible role of tRNA genes in regulating small RNA gene transcription is discussed.

Animals↗
PubMed1994

Trypanosomatid protozoa provide paradigms of eukaryotic biology.

Over the last 10 years, trypanosomatid protozoa have been the subject of intense investigation particularly focusing on their parasitic lifestyle and their intriguing and novel cell properties. These studies have furthered our understanding of the physiology and functioning of these cells and have identified a large number of biochemical and metabolic peculiarities, including mitochondrial function, enzymatic compartmentalization, and gene expression. This review focuses on the mode of gene expression and highlights areas of trypanosome research that have provided paradigms of eukaryotic biology.

Animals↗
PubMed1993

2'-O-methyl RNA oligonucleotides identify two functional elements in the trypanosome spliced leader ribonucleoprotein particle.

Using permeable trypanosomes as an in vivo model system for trans-splicing, we have searched for functional elements in the Trypanosoma brucei spliced leader (SL) RNA by masking various regions of the molecule with short antisense 2'-O-methyl RNA oligomers. Initial probing of the structure of newly synthesized SL RNA by deoxyoligonucleotide-directed ribonuclease (RNase) H cleavage revealed three accessible regions: the 5' end, sequences downstream of the 5' splice site, and a putative single-stranded sequence between stem-loops II and III, which is thought to be analogous to the mammalian Sm-binding site of U small nuclear RNAs. Using antisense 2'-O-methyl RNA oligomers, two functional elements of the SL RNA became apparent. Masking of positions 1-18 inhibited modification of the cap 4 structure of newly synthesized SL RNA and, thereby, blocked utilization of the SL RNA in trans-splicing. In addition, nucleotides +1 to +4 relative to the 5' splice site, which include the invariant GU dinucleotide were accessible to oligomer binding in the SL ribonucleoprotein particle, and their blockade resulted in complete inhibition of trans-splicing. In contrast, RNA oligomer binding to the single-stranded region between stem-loop II and III of the SL RNA had no detectable effect on trans-splicing activity of the SL RNA.

Animals↗
PubMed1993

Identification of a tRNA-like molecule that copurifies with the 7SL RNA of Trypanosoma brucei.

During the purification of Trypanosoma brucei 7SL RNA, we detected a small RNA, 76 nucleotide-long (sRNA-76), that copurified with the 7SL RNP through several different separation steps. In this study, a partial RNA sequence of sRNA-76 was obtained and a complementary oligonucleotide to the RNA sequence was used to clone the corresponding gene. sRNA-76 is very similar to a tRNA molecule and is encoded by a single copy gene. The gene is located next to a tRNA(Val) which has 75.3% homology to T. brucei tRNA(Val) that exists in a different chromosomal locus. The highest homology of sRNA-76 is to mouse and rat tRNA(Asp) (69%), to mouse tRNA(Gly) (68.1%) and to yeast suppressor tRNA(Gly) (69.5%). However, sRNA-76 is neither a tRNA(Asp) nor a tRNA(Gly), since it has a Leu anticodon. In addition, sRNA-76 deviates from the canonical tRNA structure in 3 positions. A potential for base pairing between sRNA-76 and 7SL RNA was found in the 100 nt region of 7SL RNA, which is a highly conserved region in all 7SL RNAs.

Animals↗
PubMed1993

Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts.

Many trypanosome genes are expressed as part of large polycistronic transcription units. This finding suggests that regulation of mRNA biogenesis may emphasize RNA-processing reactions more so than in other organisms. This study was undertaken to understand the temporal order of two RNA-processing reactions, trans splicing and polyadenylation, in the maturation of trypanosome mRNAs in vivo. Kinetic studies revealed rapid trans splicing of alpha-tubulin, beta-tubulin, and actin pre-mRNAs within 1 to 2 min after synthesis of the 3' splice site. Furthermore, following blockage of pre-mRNA synthesis, newly synthesized spliced leader RNA cannot be used for trans splicing, suggesting that trypanosomes do not accumulate substantial amounts of pre-mRNA which can provide splice acceptor sites. Thus, trans splicing is cotranscriptional. In addition, we show that trans splicing precedes polyadenylation in the processing of trypanosome tubulin pre-mRNAs.

Animals↗
PubMed1992

The 7SL RNA homologue of Trypanosoma brucei is closely related to mammalian 7SL RNA.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by a signal recognition particle, a small ribonucleoprotein (RNP) containing 7SL RNA. We have cloned and sequenced the gene coding for the Trypanosoma brucei 7SL RNA homologue and found that its sequence shows the highest degree of similarity to the human 7SL RNA sequence. In keeping with the prototype secondary structure of eukaryotic 7SL RNA, the trypanosome 7SL RNA secondary structure can be folded into four domains. The 7SL RNP, which sediments at approximately 11S on sucrose density gradients, was partially purified using column chromatography. A particle containing a 76-nucleotide-long RNA co-purified with the 7SL RNP; however, these particles did not co-fractionate by non-denaturing polyacrylamide gel electrophoresis.

Animals↗
PubMed1991

Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Animals↗
PubMed1991

Differences in glucose transport between blood stream and procyclic forms of Trypanosoma brucei rhodesiense.

In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitor N-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ ions and is inhibited by phlorizin, an inhibitor of Na(+)-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na(+)-dependent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23 microM) than the bloodstream carrier (apparent Km 237 microM).

Animals↗
PubMed1990

Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region.

The U2 small nuclear RNA (snRNA) of Trypanosoma brucei gambiense, a flagellated protozoon of the order Kinetoplastida, is 148 nucleotides (nt) long, and thus the smallest U2 snRNA identified so far. To examine the evolutionary conservation of this RNA among Kinetoplastida, we have cloned and sequenced the U2 genes from Trypanosoma congolense and Leishmania mexicana amazonensis, which are 145 and 141 nt in length, respectively. The sequences of the Kinetoplastida U2 snRNAs are essentially identical in the 5' half of the molecule. Surprisingly, the putative branch site recognition sequence of L. m. amazonensis U2 snRNA shows two nt changes when compared with the other two U2 snRNAs. The sequence of the 3' half of the Kinetoplastida U2 snRNAs is less conserved with T. congolense and L. m. amazonensis RNAs showing 23 and 35 nt sequence variations, respectively, when compared with the corresponding sequence of the T. b. gambiense U2 snRNA. Alignment of the flanking regions of the U2 genes revealed several elements which are conserved both in sequence and in position relative to the U2 coding region and which may function in the biosynthesis of U2 snRNAs. One upstream element specifically binds protein factor(s) present in T. brucei nuclear extracts.

Animals↗
PubMed1990

Permeable trypanosome cells as a model system for transcription and trans-splicing.

We have established conditions for Trypanosoma brucei permeable cells to study transcription and trans-splicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCl, whereas higher potassium concentrations were inhibitory. In addition, MgCl2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing. Using conditions optimal for the synthesis of the SL RNA, we observed accurate trans-splicing of newly-synthesized alpha-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced alpha-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.

Amanitins↗
PubMed1990

Destruction of U2, U4, or U6 small nuclear RNA blocks trans splicing in trypanosome cells.

We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.

Animals↗
PubMed1989

Identification and isolation of a major cell surface glycoprotein of Pneumocystis carinii.

Radioiodination of rat-derived Pneumocystis carinii obtained from an in vitro culture demonstrated the presence of a major surface glycoprotein (gp120). The glycoprotein was of the high mannose type. It exhibited adherence properties similar to those observed in the intact organism. Under nonreducing conditions, it existed as an aggregate with a molecular weight in excess of 2 x 10(6). Surface aggregating behavior and adherent quality prevented isolation of the glycoprotein by conventional methods. The glycoprotein was purified by chromatography on hydroxyapatite in the presence of sodium dodecyl sulfate under reducing conditions.

Animals↗
PubMed1988

Polygene transcripts are precursors to calmodulin mRNAs in trypanosomes.

In African trypanosomes, calmodulin is encoded by a small family of tandemly repeated genes consisting of three to four units. We show that all the members of the calmodulin cluster of Trypanosoma brucei gambiense are expressed. In addition to mature mRNAs, steady-state RNA contains a small percentage of polygene transcripts which comprise at least two and probably all calmodulin genes. The 5' ends of a portion of these molecules appear to be indistinguishable from those of mature calmodulin mRNAs. Polygene transcripts are not polyadenylated and have discrete ends which map in the intergenic regions downstream from the polyadenylation sites. Using biotinylated hybridization probes and selection of the hybrids on streptavidin-agarose, we further show that calmodulin polygene transcripts are the most abundant RNA species detected in pulse-labelled RNA of cultured procyclic trypanosomes. Our data strongly imply that polygene transcripts are authentic precursors to mature calmodulin mRNAs.

Animals↗
PubMed1986

The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes.

We have isolated the gene coding for the U2 analogue in trypanosomes. The 148 nucleotide long U2 RNA is capped and transcribed from a single copy gene. The 5' half of the molecule is highly homologous to mammalian U2 RNA, while the 3' half does not show any significant sequence homology with the mammalian counterpart. Nevertheless, the trypanosome U2 RNA can be folded into a secondary structure resembling the one proposed for U2 RNA. The presence of a U2 analogue and most likely other U RNAs in trypanosomes suggests that splicing is involved at some point in the maturation of mRNA. Possible interactions of the U2 RNA with the spliced leader RNA are considered.

Animals↗
PubMed1986

Identification of dynamic sequences in the central domain of 7SL RNA.

The human 7SL RNA component of the signal recognition particle can be separated into four major conformers by nondenaturing polyacrylamide gel electrophoresis. We have investigated what sequences give 7SL RNA the property to exist in different conformations. The human 7SL gene 7L30.1 was mutagenized using the random linker insertion approach and twelve mutant genes carrying alterations in the central domain of 7SL RNA were characterized. Mutant RNAs were produced by in vitro transcription of the various templates and their electrophoretic behaviour was determined. Bases between positions 98 and 133 as well as 206 and 251 proved to be necessary for the 7SL RNA to be able to exist in alternative conformations, while changes at the positions 85 to 97, 144 to 166 and 252 to 266 did not abolish this property. The dynamic sequences are located in the "central T" in the secondary structure of the 7SL RNA. They are phylogenetically conserved and include bases which are homologeous to 5S ribosomal RNA. A dynamic core structure composed of the dynamic parts of the 7SL RNA is suggested. An attempt was made to define the different conformers present in the wild-type 7SL RNA. These alternative configurations could play a functional role during the initial stage of protein translocation across the membrane of the endoplasmic reticulum.

Base Sequence↗