Short RNA Guides Cleavage by Eukaryotic RNase III

Lamontagne, Bruno; Elela, Sherif Abou

doi:10.1371/journal.pone.0000472

Cited by 14 publications

(24 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Their RNase III domains may interact with that of KREN1, KREN2, and KREN3 to create a catalytic site that only cleaves mRNA in the mRNA/gRNA duplex. While RNase IIIs typically cleave both strands of an RNA duplex, cleavage of one strand has been reported, and yeast Rnt1p can catalyze guide-directed cleavage of a single strand (6,7,31,48,50). Having two different proteins, i.e., KREPB4 and KREPB5, may expand the repertoire of editing sites that the editing endonucleases can recognize and cleave.…”

Section: Discussionmentioning

confidence: 99%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

106

150

View full text Add to dashboard Cite

Trypanosoma brucei has three distinct ϳ20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ϳ20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.RNA editing recodes most of the mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridine nucleotides (U's) using information provided by guide RNAs (gRNAs) (58). RNA editing is developmentally regulated and is required for parasites that cycle between insect vector and mammalian host (54). Proteins that perform catalytic steps of RNA editing have been identified: endonucleases KREN1 or KREN2 cleave at deletion or insertion sites, respectively; terminal uridylyl transferase (TUTase) KRET2 adds U's at insertion sites; U specific exoribonuclease (exoUase) KREX1 removes U's at deletion sites; and ligases KREL1 or KREL2 rejoin mRNA fragments after U addition or removal (5,13,25,30,34,55,61). These catalytic steps are coordinated by the multiprotein editosomes that sediment at ϳ20S on glycerol gradients (9, 43). KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.KREN1 and KREN2 were shown to be RNA editing endonucleases using both RNA interference (RNAi) and conditional knockout cell lines (5, 61). Both KREN1 and KREN2 are essential for the normal growth of PF and BF cells, and repression of their expression by either method led to dramatic growth defects or death, respectively. Such repression of KREN1 eliminated in vitro cleavage by ϳ20S editosomes of a deletion site in a synthetic substrate modeled on ATPase subunit 6 (A6) pre-mRNA but did not alter cleavage of an insertion site in a substrate derived from the same mRNA. These studies and other data have shown that KREN1 is an editing endonuclease with a preference for sites from which U's are deleted (29). Similarly, repression of KREN2 eliminated in vitro cleavage at insertion ed...

show abstract

Section: Discussionmentioning

confidence: 99%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

106

150

View full text Add to dashboard Cite

show abstract

“…Deletion of RNT1 perturbs the cell cycle and growth, increases the telomere length, inhibits ribosome production, impairs cell wall stress response, and induces temperature sensitivity (Catala et al, 2008). While Rnt1p normally cleaves local stem loop structure, it was shown that the enzyme could direct cleavage in trans by binding to small guide RNA in analogy to the mechanism of RNAi, which is absent in S. cerevisiae (Lamontagne and Abou Elela, 2007). …”

Section: Introductionmentioning

confidence: 99%

Structure of a Eukaryotic RNase III Postcleavage Complex Reveals a Double-Ruler Mechanism for Substrate Selection

et al. 2014

View full text Add to dashboard Cite

SUMMARY RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. The mechanism of action has been well characterized for the bacterial enzyme, but is not clear for eukaryotic RNase IIIs. Here, we describe the structure of Saccharomyces cerevisiae RNase III (Rnt1p) post-cleavage complex and explain the basis of its affinity for RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a new structural motif located at the end of Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domain function as two rulers measuring the distance between the tetraloop and the cleavage site. This unusual mechanism of substrate selectivity represents an example of the evolution of substrate selectivity and provides a framework for understanding the mechanism of action of eukaryotic RNase IIIs.

show abstract

“…Looking at the Giardia Dicer crystal structure, it is tempting to speculate that the two Dicer selected stems (39-overhang and the binding arm strand) stack together and bind to the flat linker surface in between the PAZ and the RNase domains (MacRae et al 2006) leaving the third stem protruding from the nuclease domain in space. A comparable asymmetric cleavage was also reported when the cleavages of yeast RNase III (Rnt1p) and tRNase ZL were directed in trans using guide RNA (Lamontagne and Abou Elela 2007;Nakashima et al 2007). Therefore, it is likely that this phenomenon is not specific to any given cleavage mechanism, but rather that it demonstrates the inherently limited number of three-way junction conformations that could correctly fit for catalysis.…”

Section: Discussionmentioning

confidence: 58%

“…3A,B). The position of these modifications was chosen not to affect RNA duplex formation or the overall substrate structure based on previous study using yeast and bacterial RNase III Lamontagne and Abou Elela 2007;Lavoie and Abou Elela 2008). Surprisingly, all 29-OMe substitutions were tolerated except for that located at the cleavage site (see RG-20 result).…”

Section: Selective Effects Of 29-ome On Dicer Primary Bindingmentioning

confidence: 99%

Short RNA duplexes guide sequence-dependent cleavage by human Dicer

Bergeron

Perreault²,

Elela³

2010

RNA

View full text Add to dashboard Cite

Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 39-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 39-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 29-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic.

show abstract

Short RNA Guides Cleavage by Eukaryotic RNase III

Cited by 14 publications

References 42 publications

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Structure of a Eukaryotic RNase III Postcleavage Complex Reveals a Double-Ruler Mechanism for Substrate Selection

Short RNA duplexes guide sequence-dependent cleavage by human Dicer

Contact Info

Product

Resources

About