scholarly journals Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei.

1992 ◽  
Vol 12 (5) ◽  
pp. 2043-2049 ◽  
Author(s):  
D J Koslowsky ◽  
G R Riley ◽  
J E Feagin ◽  
K Stuart
Keyword(s):  
Cytochrome Oxidase ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Developmental Regulation ◽  
Life Forms ◽  
Developmentally Regulated ◽  
Guide Rnas ◽  
Life Cycle Stages ◽  
Mitochondrial Transcripts ◽  
Atpase 6

RNA editing of several mitochondrial transcripts in Trypanosoma brucei is developmentally regulated. The cytochrome b and cytochrome oxidase II mRNAs are edited in procyclic-form parasites but are primarily unedited in bloodstream forms. The latter forms lack the mitochondrial respiratory system present in procyclic forms. Editing of the NADH dehydrogenase 7 (ND7) and ND8 transcripts is also developmentally regulated but occurs preferentially in bloodstream forms. Other transcripts, cytochrome oxidase III and ATPase 6, are edited in both life forms. We have identified many minicircle-encoded guide RNAs (gRNAs) for ATPase 6, ND7, and ND8. The characteristics of these gRNAs reveal how extensively edited RNA can be edited in the 3'-to-5' direction. Northern (RNA) blot and primer extension analyses indicate that gRNAs for transcripts whose editing is developmentally regulated are present in both procyclic and bloodstream form parasites. These results suggest that the developmental regulation of editing in these transcripts is not controlled by the presence or absence of gRNAs.

Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei

1992 ◽  
Vol 12 (5) ◽  
pp. 2043-2049
Author(s):  
D J Koslowsky ◽  
G R Riley ◽  
J E Feagin ◽  
K Stuart
Keyword(s):  
Cytochrome Oxidase ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Developmental Regulation ◽  
Life Forms ◽  
Developmentally Regulated ◽  
Guide Rnas ◽  
Life Cycle Stages ◽  
Mitochondrial Transcripts ◽  
Atpase 6

RNA editing of several mitochondrial transcripts in Trypanosoma brucei is developmentally regulated. The cytochrome b and cytochrome oxidase II mRNAs are edited in procyclic-form parasites but are primarily unedited in bloodstream forms. The latter forms lack the mitochondrial respiratory system present in procyclic forms. Editing of the NADH dehydrogenase 7 (ND7) and ND8 transcripts is also developmentally regulated but occurs preferentially in bloodstream forms. Other transcripts, cytochrome oxidase III and ATPase 6, are edited in both life forms. We have identified many minicircle-encoded guide RNAs (gRNAs) for ATPase 6, ND7, and ND8. The characteristics of these gRNAs reveal how extensively edited RNA can be edited in the 3'-to-5' direction. Northern (RNA) blot and primer extension analyses indicate that gRNAs for transcripts whose editing is developmentally regulated are present in both procyclic and bloodstream form parasites. These results suggest that the developmental regulation of editing in these transcripts is not controlled by the presence or absence of gRNAs.

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation

1991 ◽  
Vol 11 (12) ◽  
pp. 5878-5884
Author(s):  
B K Adler ◽  
M E Harris ◽  
K I Bertrand ◽  
S L Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Total Cell ◽  
Metabolic Labeling ◽  
Mechanism Of Formation ◽  
Guide Rnas ◽  
Mitochondrial Rrna ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit

1992 ◽  
Vol 12 (5) ◽  
pp. 2100-2107
Author(s):  
A E Souza ◽  
P J Myler ◽  
K Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Nadh Dehydrogenase ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Reading Frame ◽  
Guide Rnas ◽  
Iron Sulfur ◽  
Mitochondrial Dnas ◽  
A Subunit ◽  
Sulfur Protein

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.

Developmental aspects of uridine addition within mitochondrial transcripts of Trypanosoma brucei

1988 ◽  
Vol 8 (3) ◽  
pp. 1259-1265
Author(s):  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome Oxidase ◽  
Trypanosoma Brucei ◽  
Cytochrome B ◽  
Respiratory System ◽  
Life Cycle Stage ◽  
Stage Specificity ◽  
Life Cycle Stages ◽  
Mitochondrial Transcripts ◽  
Cytochrome Oxidase Ii

The mitochondrial respiratory system is absent in slender bloodstream forms of Trypanosoma brucei, incomplete in stumpy bloodstream forms, and complete in procyclic (insect) forms. The steady-state abundance of transcripts of some mitochondrially encoded components of the respiratory system correlates with its differential expression in different life cycle stages. Recently, it was reported that uridines which are not encoded in the genome are added to cytochrome b and cytochrome oxidase II transcripts. We now report that the (U)+ transcripts of both genes are found in procyclic forms and to some degree in stumpy forms but are absent in slender forms. The uridine additions to cytochrome oxidase II correct a frameshift in the gene and presumably allow production of a full-length protein, whereas those added to cytochrome b create an in-frame AUG which extends the N terminus of the predicted protein by 20 amino acids. The stage specificity of uridine additions to these transcripts thus reflects the life cycle stage during which the protein products would be used. Transcripts of MURF2, a gene of unknown function, have additional uridines in both slender and procyclic forms which create two in-frame AUGs. MURF2 transcripts additionally differ from the DNA sequence in ways which cannot be explained by uridine addition alone, implying that other processes alter these transcripts.

scholarly journals Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit.

1992 ◽  
Vol 12 (5) ◽  
pp. 2100-2107 ◽  
Author(s):  
A E Souza ◽  
P J Myler ◽  
K Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Nadh Dehydrogenase ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Reading Frame ◽  
Guide Rnas ◽  
Iron Sulfur ◽  
Mitochondrial Dnas ◽  
A Subunit ◽  
Sulfur Protein

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.

scholarly journals Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation.

1991 ◽  
Vol 11 (12) ◽  
pp. 5878-5884 ◽  
Author(s):  
B K Adler ◽  
M E Harris ◽  
K I Bertrand ◽  
S L Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Total Cell ◽  
Metabolic Labeling ◽  
Mechanism Of Formation ◽  
Guide Rnas ◽  
Mitochondrial Rrna ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

scholarly journals Developmental aspects of uridine addition within mitochondrial transcripts of Trypanosoma brucei.

1988 ◽  
Vol 8 (3) ◽  
pp. 1259-1265 ◽  
Author(s):  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome Oxidase ◽  
Trypanosoma Brucei ◽  
Cytochrome B ◽  
Respiratory System ◽  
Life Cycle Stage ◽  
Stage Specificity ◽  
Life Cycle Stages ◽  
Mitochondrial Transcripts ◽  
Cytochrome Oxidase Ii

The mitochondrial respiratory system is absent in slender bloodstream forms of Trypanosoma brucei, incomplete in stumpy bloodstream forms, and complete in procyclic (insect) forms. The steady-state abundance of transcripts of some mitochondrially encoded components of the respiratory system correlates with its differential expression in different life cycle stages. Recently, it was reported that uridines which are not encoded in the genome are added to cytochrome b and cytochrome oxidase II transcripts. We now report that the (U)+ transcripts of both genes are found in procyclic forms and to some degree in stumpy forms but are absent in slender forms. The uridine additions to cytochrome oxidase II correct a frameshift in the gene and presumably allow production of a full-length protein, whereas those added to cytochrome b create an in-frame AUG which extends the N terminus of the predicted protein by 20 amino acids. The stage specificity of uridine additions to these transcripts thus reflects the life cycle stage during which the protein products would be used. Transcripts of MURF2, a gene of unknown function, have additional uridines in both slender and procyclic forms which create two in-frame AUGs. MURF2 transcripts additionally differ from the DNA sequence in ways which cannot be explained by uridine addition alone, implying that other processes alter these transcripts.

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei

1985 ◽  
Vol 5 (11) ◽  
pp. 3041-3047
Author(s):  
D P Jasmer ◽  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Trypanosoma Brucei ◽  
Protein Coding ◽  
Oxidase Subunit ◽  
Life Cycle Stages ◽  
Multiple Processes ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

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