Guide RNAs in kinetoplastid mitochondria have a nonencoded 3′ oligo(U) tail involved in recognition of the preedited region

Blum, Beat; Simpson, Larry

doi:10.1016/0092-8674(90)90375-o

Cited by 225 publications

(158 citation statements)

References 22 publications

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“…PhosphorImager analysis of the lanes corresponding to 72 h of RET1 RNAi induction shows that in the control tetracycline (Ϫtet) lane Ϸ90% of the radioactivity is located in the region indicated as gRNAs (Fig. 4C), and only 10% corresponds to the region with molecules shortened by 1-20 bases, which covers the average length of the oligo[U] tail (19). Down-regulation of RET1 expression shifted this balance to Ϸ60% of gRNAs having shortened oligo[U] tails.…”

Section: Stability Of L-complex Is Unaffected By Down-regulation Of Rmentioning

confidence: 99%

A tale of two TUTases

Aphasizhev

Aphasizheva

Simpson

2003

Proc. Natl. Acad. Sci. U.S.A.

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103

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The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3 terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3 oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purificationtagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3 end of a single-stranded RNA, thereby confirming that RET2 is a 3 TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro Uinsertion editing and has no effect on the length of the 3 oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3 end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.U -insertion͞deletion RNA editing in trypanosome mitochondria involves the annealing of a guide RNA (gRNA) to an mRNA, endonuclease cleavage at a non-base-paired editing site adjacent to the RNA duplex, addition or deletion of Us to or from the 3Ј end of the 5Ј cleavage fragment, and religation of the two mRNA fragments (1-4). Each gRNA mediates the editing of 1-5 sites in a 3Ј to 5Ј polarity, and multiple overlapping gRNAs mediate the editing of an entire domain, also in a 3Ј to 5Ј polarity (5).We previously isolated the RNA editing terminal uridylyl transferase (TUTase) 1 (RET1) and showed it to be present both as a free tetramer and as a component of a high molecular weight complex (TUT II) (6). This complex migrates on a native gel at Ϸ700 kDa and interacts in an RNase-sensitive manner with the larger ligase-containing complex (L-complex) (7). Downregulation of RET1 expression in Trypanosoma brucei by conditional RNA interference (RNAi) caused a decrease in the steady-state abundance of edited mRNAs without any effect on transcription (6). The core L-complex and associated proteins, capable of both U-insertion and U-deletion editing activity in vitro, was isolated from Leishmania tarentolae mitochondrial extract by tandem affinity purification (TAP) (8), followed by glycerol gradient sedimentation (7). The L-complex contains two adenylatable RNA ligases, REL1 and REL2, three related zinc finger-containing proteins, two proteins with RNase III motifs, two proteins with an AP endonuclease exonuclease phosphatase motif, a se...

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Section: Stability Of L-complex Is Unaffected By Down-regulation Of Rmentioning

confidence: 99%

A tale of two TUTases

Aphasizhev

Aphasizheva

Simpson

2003

Proc. Natl. Acad. Sci. U.S.A.

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103

181

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“…RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases.…”

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confidence: 99%

The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra-and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.cross-linking | proteomics | Trypanosoma brucei | RNA editing | editosome T he kinetoplastid Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in subSaharan Africa. Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA, which is composed of interlocked DNA maxicircles and minicircles (1, 2). Several identical maxicircles encode two ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing (3-9). RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).There are three similar versions of ∼20S editosomes that have a common set of 12 proteins (SI Appendix, Fig. S1 and Table S1). However, they are compositionally and functionally distinct in that each...

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“…The information for selection of editing sites resides in small "guide" RNAs that are mainly encoded in the kinetoplast DNA minicircles (7,14). These RNA molecules have 3Ј oligo-U tails (15), which are added posttranscriptionally by the RET1 TUTase (16,17). Down-regulation of RET1 expression by RNA interference (RNAi) results in a decrease in the steady-state abundance of edited transcripts (16) because of an effect on the 3Ј oligo-U tail of the guide RNAs (37).…”

mentioning

confidence: 99%

The Effect of RNA Interference Down-regulation of RNA Editing 3′-Terminal Uridylyl Transferase (TUTase) 1 on Mitochondrial de Novo Protein Synthesis and Stability of Respiratory Complexes in Trypanosoma brucei

Neboháčová

Maslov

Falick

et al. 2004

Journal of Biological Chemistry

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Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F 1 ⅐F 0 -ATPase complex and on several other mitochondrial proteins.Gene expression in the kinetoplast-mitochondrion of trypanosomatid protists involves a unique post-transcriptional mRNA maturation process, termed RNA editing, whereby Uresidues are inserted to or deleted from a pre-edited transcript, and a translatable reading frame is thus created (1-5). 12 of the 18 protein-coding genes in the maxicircle component of the mitochondrial or kinetoplast DNA in Trypanosoma brucei or Leishmania tarentolae encode transcripts that require varying degrees of editing for translation competence (6). These include mRNAs for apocytochrome b (Cyb), 1 subunits II and III of cytochrome c oxidase (COII and COIII, respectively), subunit 6 of ATPase (6), several subunits of NADH dehydrogenase, and a few others. Some transcripts, such as the cytochrome oxidase subunit I (COI) mRNA, do not require editing for translation. The mechanism of editing includes cleavage of RNA at specific editing sites, and the addition or deletion of a defined number of U-residues followed by religation (7,8). The reactions are performed by large protein complexes which include a 3Ј terminal uridylyl transferase (TUTase), an RNA ligase, endo-and exonuclease activities, as well as several additional auxiliary factors of undefined function (9 -12). Subcomplexes exist that contain subsets of proteins and which may specialize in Uaddition or U-deletion (13). The information for selection of editing sites resides in small "guide" RNAs that are mainly encoded in the kinetoplast DNA minicircles (7, 14). These RNA molecules have 3Ј oligo-U tails (15), which are added posttranscriptionally by the RET1 TUTase (16, 17). Down-regulation of RET1 expression by RNA interference (RNAi) results in a decrease in the steady-state abundance of edited transcripts (16) because of an effect on the 3Ј oligo-U tail of the guide RNAs (37).Although editing provides translatable transcripts, inhibition of editing should affect protein synthesis and, consequently, the assembly of respiratory complexes in the kinetoplast. The de novo synthesis of COI and Cyb po...

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Guide RNAs in kinetoplastid mitochondria have a nonencoded 3′ oligo(U) tail involved in recognition of the preedited region

Cited by 225 publications

References 22 publications

A tale of two TUTases

A tale of two TUTases

The Architecture of Trypanosoma brucei editosomes

The Effect of RNA Interference Down-regulation of RNA Editing 3′-Terminal Uridylyl Transferase (TUTase) 1 on Mitochondrial de Novo Protein Synthesis and Stability of Respiratory Complexes in Trypanosoma brucei

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