Crystal Structures of T. brucei MRP1/MRP2 Guide-RNA Binding Complex Reveal RNA Matchmaking Mechanism

Schumacher, Maria A.; Karamooz, Elham; Zı́ková, Alena; Trantı́rek, Lukáš; Lukeš, Julius

doi:10.1016/j.cell.2006.06.047

Cited by 99 publications

(122 citation statements)

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“…32,33 All oU-RNA sequences can be folded into structures consisting of two stem loops as observed for multiple gRNAs. [7][8][9] The calculated ΔG values range between -4.2 to -9.9 kcal/ mol, consistent with the observed Gibbs free energy of canonical gRNAs. 7 Expression analysis of novel oU-RNAs.…”

Section: © 2 0 0 8 L a N D E S B I O S C I E N C E D O N O T D I S supporting

confidence: 62%

“…7 The molecules are 3' oligouridylated and adopt a common secondary (2D) structure, which consists of two hairpin elements. [7][8][9] This structure becomes progressively unfolded as the editing reaction proceeds, ultimately generating a fully basepaired gRNA/mRNA hybrid molecule. In these gRNA/ mRNA hybrids, canonical as well as non Watson/Crick-type base pairs can be found.…”

Section: Introductionmentioning

confidence: 99%

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Identification of novel guide RNAs from the mitochondria ofTrypanosoma brucei

Madej¹,

Niemann²,

Hüttenhofer³

et al. 2008

RNA Biology

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The majority of mitochondrial mRNAs in African trypanosomes are subject to an RNA editing reaction, which is characterized by the insertion and/or deletion of U nucleotides only. The reaction creates functional mRNAs and is catalyzed by a high molecular mass enzyme complex, the editosome. Editosomes interact with a unique class of small non-coding, 3'-oligouridylated (oU) RNAs, so-called guide RNAs (gRNAs). Guide RNAs function as transacting templates in the U deletion/insertion reaction and thus, represent key components in the reaction cycle. Furthermore, by utilizing different gRNAs, alternative editing events can take place, thereby expanding the protein diversity in the mitochondria of the parasites. In this study, we have analyzed small, non-coding mitochondrial transcripts from Trypanosoma brucei. By generating cDNA libraries from size-selected RNA populations we identified 51 novel oU-RNAs. For 29 of these RNAs we were able to predict cognate mRNA targets. By Northern blot analysis, we verified the expression of 22 of these oU-RNAs and demonstrate that they share all known gRNA characteristics. Five of these 51 putative gRNAs are characterized by single mismatches to their cognate, fully edited mRNA sequences suggesting that they could act as gRNAs for alternative editing events.

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Section: © 2 0 0 8 L a N D E S B I O S C I E N C E D O N O T D I S supporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Identification of novel guide RNAs from the mitochondria ofTrypanosoma brucei

Madej¹,

Niemann²,

Hüttenhofer³

et al. 2008

RNA Biology

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“…The final column summarizes the demonstrated (and proposed) roles in editing, as cited in the Introduction and the above references. mHel61p helicase of the z20S complex may aid removal of gRNAs after editing (Missel et al 1997); also proteins separate from the z20S complex can affect editing, including TbMP108/KRET1 (that adds U-tails onto gRNAs) (Aphasizhev et al 2002(Aphasizhev et al , 2003c, TbgBP21 and TbgBP25 (that stimulate RNA annealing) (Müller et al 2001;Müller and Göringer 2002;Aphasizhev et al 2003b;Schumacher et al 2006), REAP1 (a putative mRNA-binding protein) , and RBP16 (a CYb RNA factor) (Pelletier and Read 2003). …”

Section: Introductionmentioning

confidence: 99%

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

Law

O’Hearn

Sollner‐Webb³

2008

RNA

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Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by ;20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 39-U-exonuclease [39-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 39-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.

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“…This complex catalyzes matchmaker RNA/RNA annealing, i.e. basepairing followed by dissociation of the RNA duplex from the proteins (11)(12)(13). RBP16, another trypanosomal mitochondrial RNA-binding protein, has also been shown to facilitate gRNA/pre-mRNA annealing in vitro (14,15).…”

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confidence: 99%

“…This provides for the successive use of the gRNAs. Association of the mRNAs and gRNAs may be mediated by the MRP complex, which consists of two copies each of two related proteins (11). This complex catalyzes matchmaker RNA/RNA annealing, i.e.…”

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confidence: 99%

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

Schnaufer

Park

et al. 2010

Journal of Biological Chemistry

126

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Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ϳ20S on glycerol gradients. These ϳ20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ϳ20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ϳ20S editosomes during the editing process.

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Crystal Structures of T. brucei MRP1/MRP2 Guide-RNA Binding Complex Reveal RNA Matchmaking Mechanism

Cited by 99 publications

References 49 publications

Identification of novel guide RNAs from the mitochondria ofTrypanosoma brucei

Identification of novel guide RNAs from the mitochondria ofTrypanosoma brucei

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

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