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

1993 ◽  
Vol 13 (1) ◽  
pp. 720-725
Author(s):  
E Ullu ◽  
K R Matthews ◽  
C Tschudi
Keyword(s):  
Rna Processing ◽  
Temporal Order ◽  
Kinetic Studies ◽  
Splice Acceptor ◽  
Mrna Synthesis ◽  
Alpha Tubulin ◽  
Spliced Leader ◽  
Polycistronic Transcription ◽  
Trans Splicing

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.

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

1993 ◽  
Vol 13 (1) ◽  
pp. 720-725 ◽  
Author(s):  
E Ullu ◽  
K R Matthews ◽  
C Tschudi
Keyword(s):  
Rna Processing ◽  
Temporal Order ◽  
Kinetic Studies ◽  
Splice Acceptor ◽  
Mrna Synthesis ◽  
Alpha Tubulin ◽  
Spliced Leader ◽  
Polycistronic Transcription ◽  
Trans Splicing

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.

scholarly journals An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly

2017 ◽  
Vol 45 (14) ◽  
pp. 8474-8483 ◽  
Author(s):  
Lucas Philippe ◽  
George C. Pandarakalam ◽  
Rotimi Fasimoye ◽  
Neale Harrison ◽  
Bernadette Connolly ◽  
...  
Keyword(s):  
Crucial Role ◽  
De Novo ◽  
Genetic Screen ◽  
Spliced Leader ◽  
Trans Splicing

Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus

1991 ◽  
Vol 11 (6) ◽  
pp. 3180-3190
Author(s):  
J Huang ◽  
L H van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Processing ◽  
Rnase A ◽  
Chain Elongation ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Transcriptional Elongation ◽  
Splice Acceptor ◽  
Trans Splicing ◽  
Nascent Rna

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

scholarly journals Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus.

1991 ◽  
Vol 11 (6) ◽  
pp. 3180-3190 ◽  
Author(s):  
J Huang ◽  
L H van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Processing ◽  
Rnase A ◽  
Chain Elongation ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Transcriptional Elongation ◽  
Splice Acceptor ◽  
Trans Splicing ◽  
Nascent Rna

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo

1992 ◽  
Vol 12 (11) ◽  
pp. 4844-4851
Author(s):  
K P McNally ◽  
N Agabian
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Splicing ◽  
Rna Modification ◽  
Ribonucleoprotein Particle ◽  
Rna Methylation ◽  
Spliced Leader ◽  
The Core ◽  
Trans Splicing ◽  
In Trans

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection

1988 ◽  
Vol 8 (3) ◽  
pp. 1352-1360
Author(s):  
R E Layden ◽  
H Eisen
Keyword(s):  
Site Selection ◽  
Surface Glycoprotein ◽  
Splice Sites ◽  
Splice Acceptor ◽  
Splice Site Selection ◽  
Primary Transcript ◽  
Spliced Leader ◽  
Trans Splicing ◽  
Multiple Messages ◽  
Trypanosoma Equiperdum

We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

scholarly journals Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

1996 ◽  
Vol 15 (16) ◽  
pp. 4380-4391 ◽  
Author(s):  
S. Lücke ◽  
G. L. Xu ◽  
Z. Palfi ◽  
M. Cross ◽  
V. Bellofatto ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Spliced Leader ◽  
Trans Splicing ◽  
Spliced Leader Rna

scholarly journals Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

1992 ◽  
Vol 12 (11) ◽  
pp. 4844-4851 ◽  
Author(s):  
K P McNally ◽  
N Agabian
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Splicing ◽  
Rna Modification ◽  
Ribonucleoprotein Particle ◽  
Rna Methylation ◽  
Spliced Leader ◽  
The Core ◽  
Trans Splicing ◽  
In Trans

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

scholarly journals Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

1988 ◽  
Vol 8 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
R E Layden ◽  
H Eisen
Keyword(s):  
Site Selection ◽  
Surface Glycoprotein ◽  
Splice Sites ◽  
Splice Acceptor ◽  
Splice Site Selection ◽  
Primary Transcript ◽  
Spliced Leader ◽  
Trans Splicing ◽  
Multiple Messages ◽  
Trypanosoma Equiperdum

We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Sign in / Sign up

Export Citation Format

Share Document