Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21

Müller, Ulrich F.; Lambert, Laurence; Göringer, H. Ulrich

doi:10.1093/emboj/20.6.1394

Cited by 87 publications

(147 citation statements)

References 45 publications

(72 reference statements)

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“…However, the Kd values were significantly lower for both the native and the reconstructed complexes, indicating a higher affinity of binding of the complex than either of the component proteins. The Kd for the binding of Ltp28 with single-stranded RNA was approximately 10-fold higher than that reported for the binding of T. brucei gBP21 to single-stranded RNA (Muller et al 2001), which may reflect a species variation or differences in the recombinant protein preparation. However, the Kd of binding of the Ltp26/Ltp28 complex to single-stranded RNA (9.6 nM) is nearly identical to that of gBP21 (Muller et al 2001).…”

Section: Rna-binding Properties Of the Purified And Reconstituted Ltpcontrasting

confidence: 54%

“…The RNA-binding mitochondrial protein gBP21, from T. brucei, has been extensively studied (Köller et al 1994;Allen et al 1998;Lambert et al 1999;Muller et al 2001) and has several properties that make it a very likely candidate for indirect involvement in editing. These involve high-affinity binding to gRNA and coimmunoprecipitation with in vitro editing activity.…”

Section: Introductionmentioning

confidence: 99%

“…These involve high-affinity binding to gRNA and coimmunoprecipitation with in vitro editing activity. An RNA annealing activity was also demonstrated, and it was proposed that this protein played a role in the initial interaction of mRNA and the cognate gRNA (Muller et al 2001). However, a targeted knockout of the corresponding gene had no apparent affect on editing in vivo (Lambert et al 1999).…”

Section: Introductionmentioning

confidence: 99%

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A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

Aphasizhev¹,

Aphasizheva²,

Nelson³

et al. 2003

RNA

101

121

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A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3 TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

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Section: Rna-binding Properties Of the Purified And Reconstituted Ltpcontrasting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

Aphasizhev¹,

Aphasizheva²,

Nelson³

et al. 2003

RNA

101

121

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“…It may be that RNA editing of developmentally regulated transcripts requires the participation of specific protein factors that allow efficient gRNA/mRNA annealing. A number of gRNA binding proteins have been characterized that accelerate the hybridization of cognate guide RNA/pre-edited mRNA pairs [43][44][45][46]. Recently, Pelletier and Read [46] showed that RNAi knockdowns of the Y-box protein, RBP16, led to a 98% reduction in the levels of edited CYb mRNA.…”

Section: Discussionmentioning

confidence: 99%

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

Koslowsky¹,

Reifur²,

Yu³

et al. 2004

RNA Biology

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The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K D in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

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“…The mitochondrial RNA-binding protein complex contains two small RNA-binding proteins and three associated proteins, and may possibly be involved in the initial annealing of the guide RNA and the mRNA (4)(5)(6)(7)(8)(9). The RNAediting terminal uridylyl transferase 1 (TUTase 1) (RET1) complex contains the RET1 tetramer and one or more unidentified components (10).…”

mentioning

confidence: 99%

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Gao

Simpson

Kang

et al. 2005

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

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The Ϸ20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains Ϸ16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.editing ͉ RNA-editing ligase 1 ͉ RNA-editing ligase 2 ͉ trypanosome S everal macromolecular complexes have been identified that are involved in U-insertion͞deletion RNA editing in trypanosomatid mitochondria (1-3). The mitochondrial RNA-binding protein complex contains two small RNA-binding proteins and three associated proteins, and may possibly be involved in the initial annealing of the guide RNA and the mRNA (4-9). The RNAediting terminal uridylyl transferase 1 (TUTase 1) (RET1) complex contains the RET1 tetramer and one or more unidentified components (10). RET1 has been shown to be involved in the addition of nonencoded Us to the 3Ј ends of guide RNAs (11). The RNA ligase-containing L-complex was initially detected as an ␣-32 P-ATP-labeled particle sedimenting Ϸ20S in glycerol gradients and migrating as a single band with an approximate size of 1,200-2,000 kDa in native gels (12,13). This complex has been labeled the editosome but it seems appropriate to reserve this nomenclature for the entire RNA͞protein supercomplex once it is more defined. The L-complex has been isolated from Trypanosoma brucei and Leishmania tarentolae mitochondria by column chromatography (13-16) and by tandem affinity purification (TAP) chromatography (17). At least 16 protein components have been identified, including the two RNA-editing ligases (RELs) (REL1 and REL2), a second 3Ј TUTase (RET2), three to four RNase III-motif proteins, three related zinc finger-motif proteins, two AP-endo-exonucleasephosphatase-motif proteins, two RNA binding-motif proteins, and several proteins lacking identifiable motifs.The two ligases are localized in subcomplexes that have been partially defined by coimmunoprecipitation experiments and...

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Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21

Cited by 87 publications

References 45 publications

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

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