Molecular architecture of the human tRNA ligase complex

Kroupova, Alena; Ackle, Fabian; Asanović, Igor; Weitzer, Stefan; Boneberg, Franziska M; Faini, Marco; Leitner, Alexander; Chui, Alessia; Aebersold, Ruedi; Martı́nez, Javier; Jínek, Martin

doi:10.7554/elife.71656

Cited by 25 publications

(56 citation statements)

References 99 publications

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“…In this study, we focused on the RTCB ligase complex as a candidate molecule to regulate stress-induced tiRNA production. Human RTCB ligase complex (RTCB-LC) is an RNA ligase complex comprising of at least four core subunits, RTCB, DDX1, FAM98B and CGI-99 [ 34 ]. While RTCB is the only subunit essential for the ligation reaction, it requires Archease (ARCH) as a cofactor for multiple turnover reactions [ 35 ].…”

Section: Introductionmentioning

confidence: 99%

RTCB Complex Regulates Stress-Induced tRNA Cleavage

Akiyama

Takenaka

Kasahara

et al. 2022

IJMS

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Under stress conditions, transfer RNAs (tRNAs) are cleaved by stress-responsive RNases such as angiogenin, generating tRNA-derived RNAs called tiRNAs. As tiRNAs contribute to cytoprotection through inhibition of translation and prevention of apoptosis, the regulation of tiRNA production is critical for cellular stress response. Here, we show that RTCB ligase complex (RTCB-LC), an RNA ligase complex involved in endoplasmic reticulum (ER) stress response and precursor tRNA splicing, negatively regulates stress-induced tiRNA production. Knockdown of RTCB significantly increased stress-induced tiRNA production, suggesting that RTCB-LC negatively regulates tiRNA production. Gel-purified tiRNAs were repaired to full-length tRNAs by RtcB in vitro, suggesting that RTCB-LC can generate full length tRNAs from tiRNAs. As RTCB-LC is inhibited under oxidative stress, we further investigated whether tiRNA production is promoted through the inhibition of RTCB-LC under oxidative stress. Although hydrogen peroxide (H2O2) itself did not induce tiRNA production, it rapidly boosted tiRNA production under the condition where stress-responsive RNases are activated. We propose a model of stress-induced tiRNA production consisting of two factors, a trigger and booster. This RTCB-LC-mediated boosting mechanism may contribute to the effective stress response in the cell.

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Section: Introductionmentioning

confidence: 99%

RTCB Complex Regulates Stress-Induced tRNA Cleavage

Akiyama

Takenaka

Kasahara

et al. 2022

IJMS

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“…Stress-induced tRNA cleavage at the anticodon loop is thought to be an irreversible process. Nevertheless, it should be considered that most of the enzymatic activities used in this work to repair anticodon-cleaved nicked tRNAs are present in human cells (Kroupova et al, 2021; Pinto et al, 2020). Reversibility of stress-induced nicked tRNA formation in cellulo arises as an exciting possibility.…”

Section: Discussionmentioning

confidence: 99%

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Costa

Calzi

Castellano

et al. 2022

Preprint

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Cells can release ribosomes and full-length tRNAs into the extracellular space, but these species are rapidly processed by extracellular RNases into stable rRNA and tRNA-derived fragments (tDRs). These nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their function or biomarker potential. The high extracellular stability of most of these fragments is also mechanistically unclear. To interrogate this, we measured the stability of several naked RNAs when incubated in human serum, urine and CSF. Interestingly, we identified extracellularly-produced tDRs with half-lives of several hours in human biofluids. Contrary to widespread assumptions, these intrinsically stable tRNA halves are mostly not in fragment form. On the contrary, they are present as full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs), as supported by native electrophoresis. Standard molecular biology techniques, including phenol-base RNA extraction, induce the denaturation of nicked tRNAs and the release of tDRs. To demonstrate this by an alternative approach, we performed enzymatic repair of nicked tRNAs with either RtcB or T4 PNK and Rnl1, obtaining a single tRNA-sized northern blot band that was not produced if samples were heat-denatured before enzymatic repair. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions. These protocols were used to identify nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Additionally, we showed that dissociation of nicked tRNAs produces single-stranded tRNA halves that can be spontaneously taken up by human epithelial cells. Overall, this study builds a framework for investigating potential intercellular communication pathways mediated by stable nv-exRNAs. Moreover, we provide useful methods to uncover a hidden layer of the extracellular RNAome composed of intrinsically stable, nick-containing, highly structured RNAs.

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“…While the excision of the intron appears to be conserved across eukaryotes, the ligation pathways diverge from yeast and metazoans (Figure 1). In humans, the cleaved exons are directly re‐sealed by the RTCB tRNA ligase complex which is a large multi‐protein complex containing the ligase RTCB (sometimes referred to as HSPC117) along with several additional cofactors including archease, the DEAD‐box helicase DDX1, ASW(C2orf49), FAM98B, and CGI‐99 (Kroupova et al, 2021; Popow et al, 2011, 2014). In addition to sealing the exons halves together, RTCB can also drive the circularization of tRNA introns (Schmidt et al, 2019).…”

Section: Overview Of Transfer Rna Splicing In Eukaryotesmentioning

confidence: 99%

Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

2022

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The splicing of transfer RNA (tRNA) introns is a critical step of tRNA maturation, for intron-containing tRNAs. In eukaryotes, tRNA splicing is a multi-step process that relies on several RNA processing enzymes to facilitate intron removal and exon ligation. Splicing is initiated by the tRNA splicing endonuclease (TSEN) complex which catalyzes the excision of the intron through its two nuclease subunits. Mutations in all four subunits of the TSEN complex are linked to a family of neurodegenerative and neurodevelopmental diseases known as pontocerebellar hypoplasia (PCH). Recent studies provide molecular insights into the structure, function, and regulation of the eukaryotic TSEN complex and are beginning to illuminate how mutations in the TSEN complex lead to neurodegenerative disease. Using new advancements in the prediction of protein structure, we created a three-dimensional model of the human TSEN complex. We review functions of the TSEN complex beyond tRNA splicing by highlighting recently identified substrates of the eukaryotic TSEN complex and discuss mechanisms for the regulation of tRNA splicing, by enzymes that modify cleaved tRNA exons and introns. Finally, we review recent biochemical and animal models that have worked to address the mechanisms that drive PCH and synthesize these studies with previous studies to try to better understand PCH pathogenesis.

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Molecular architecture of the human tRNA ligase complex

Cited by 25 publications

References 99 publications

RTCB Complex Regulates Stress-Induced tRNA Cleavage

RTCB Complex Regulates Stress-Induced tRNA Cleavage

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

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