Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

Wu, Jingyan; Hopper, Anita K.

doi:10.1101/gad.244673.114

Cited by 37 publications

(54 citation statements)

References 40 publications

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“…RTCB not only is responsible for tRNA ligation, but it is also responsible for creation of tricRNAs in metazoans and archaea (22,57,64,65). Both metazoans and archaea have been shown to have produce tricRNAs, whereas yeast introns are primarily linear and rapidly degraded (66,67). Because circularized introns are very stable (57), CLP1 may play a critical role in circular RNA homeostasis by inhibiting RTCB-mediated ligation and acting as negative regulator of tricRNA biogenesis.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Hayne

Schmidt²,

Matera³

et al. 2019

Preprint

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The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.We thank Drs. Andrew Sikkema and Marcos Morgan as well as members of the Stanley Lab for their critical reading of this manuscript. We would like to acknowledge Robert Petrovich and the NIEHS Protein Expression Facility for assistance with protein expression in HEK293 suspension cultures and for supplying the anti-GFP resin. We also thank Robert Dutcher for his assistance with running SEC-MALS.

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Section: Discussionmentioning

confidence: 99%

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Hayne

Schmidt²,

Matera³

et al. 2019

Preprint

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“…While it is unclear if the 2’, 3’-cyclic phosphate 3’ end of the 7S precursor is acted upon by a phosphodiesterase or other enzyme prior to degradation, our structural model predicts that a 2’, 3’-cyclic phosphate 3’ end could be accommodated in the Rrp44 active site. Previous studies implicated the 5’-3’ decay pathway in degradation of 2’, 3’-cyclic phosphate tRNA introns (Wu and Hopper, 2014), but the observation that Rrp44 and related human enzymes can degrade 3’ phosphorylated RNA begs the question as to whether the RNA exosome also participates in the degradation of aberrant 3’ modified forms of RNA, or if this activity contributes to generating clean 3’OH RNA ends to promote post-transcriptional polyadenylation by TRAMP (LaCava et al, 2005; Wyers et al, 2005) or 3’ trimming by Rrp6.…”

Section: Discussionmentioning

confidence: 99%

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

2016

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SUMMARY The eukaryotic RNA exosome is an essential and conserved 3’ to 5’ exoribonuclease complex that degrades or processes nearly every class of cellular RNA. The nuclear RNA exosome includes a nine-subunit non-catalytic core that binds Rrp44 (Dis3) and Rrp6 subunits to modulate their processive and distributive 3’ to 5’ exoribonuclease activities, respectively. Here, we utilize an engineered RNA with two 3’ ends to obtain a crystal structure of an eleven-subunit nuclear exosome bound to RNA at 3.1 Å. The structure reveals an extended RNA path to Rrp6 that penetrates into the non-catalytic core, contacts between the non-catalytic core and Rrp44 that inhibit exoribonuclease activity, and features of the Rrp44 exoribonuclease site that support its ability to degrade 3’ phosphate RNA substrates. Using reconstituted exosome complexes, we show that 3’ phosphate RNA is not a substrate for Rrp6, but is readily degraded by Rrp44 in the nuclear exosome.

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“…For human cell culture, HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin (Gibco) at 37° and 5% CO2. Cells (2 x 10 6 ) were plated in T25 flasks and transiently transfected with 2.5 µg plasmid DNA per flask using FuGENE HD transfection reagent (Promega) according to the manufacturer's protocol. Cells were harvested 72 hours post-transfection.…”

Section: Cell Culture and Transfectionsmentioning

confidence: 99%

“…A separate 2'-phosphotransferase enzyme called Tpt1 removes the remaining 2'-phosphate at the junction of the newly formed tRNA. The intron, now phosphorylated on its 5' end, is degraded by a 5' to 3' exonuclease, creating a supply of nucleotides (6).…”

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confidence: 99%

Molecular determinants of metazoan tricRNA biogenesis

Schmidt

Giusto

Matera

2018

Preprint

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Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in both human and fly cells.We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient.Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identify trans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players in Drosophila tricRNA biogenesis.

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Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

Cited by 37 publications

References 40 publications

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

Molecular determinants of metazoan tricRNA biogenesis

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