The Making and Breaking of tRNAs by Ribonucleases

Abstract

Ribonucleases (RNases) play important roles in supporting canonical and non-canonical roles of tRNAs by catalyzing the cleavage of the tRNA phosphodiester backbone. Here, we highlight how recent advances in cryo-electron microscopy (cryo-EM), protein structure prediction, reconstitution experiments, tRNA sequencing, and other studies have revealed new insight into the nucleases that process tRNA. This represents a very diverse group of nucleases that utilize distinct mechanisms to recognize and cleave tRNA during different stages of a tRNA’s life cycle including biogenesis, fragmentation, surveillance, and decay. In this review, we provide a synthesis of the structure, mechanism, regulation, and modes of tRNA recognition by tRNA nucleases, along with open questions for future investigation.

Ribonucleases mediate the life cycle of tRNAs

Transfer RNAs (tRNAs) play a fundamental role in protein synthesis, delivering amino acids as they translate mRNA codons on the ribosome. Beyond their essential role in translation, new non-canonical roles for tRNAs are continuing to be discovered[1]. Most eukaryotic tRNAs are composed of 73–100 nucleotides that form the classic tRNA cloverleaf secondary structure (Fig. 1a). tRNAs fold into an L-shaped tertiary structure with the acceptor stem, the site of amino acid attachment, at one end and the anticodon stem loop at the other. Dysregulation of tRNAs is associated with many human diseases including neurodevelopmental and neurodegenerative diseases, and several human cancers, underscoring the need to understand the major factors involved in tRNA regulation[2–5].

Figure 1. Overview of tRNA structure and protein RNase cleavage mechanisms.

(A) Secondary (left) and crystal structure (right) of Saccharomyces cerevisiae tRNA-Phe (PDB ID 1EHZ [116]). The tRNA cloverleaf secondary structure is composed of the acceptor stem (cyan), D-Loop (pink), anticodon stem (orange), variable region (gold), and pseudouridine and cytidine-containing (TψC or T) loop (green). The position of the discriminator base (dark blue) and anticodon (dark red) are also indicated. (B) Protein RNases that utilize metal ions generate nucleolytic cleavage products with 5′-phosphate and 3′-hydroxyl ends. The hydrolysis of the phosphodiester bond relies on the coordination of one or more metal ions in the nuclease active site. (C) Metal independent protein RNases mediate the attack of the 2′ hydroxyl on the phosphate, generating cleavage products containing 2′3′-cyclic phosphate and 5′-hydroxyl ends. Transesterification reactions are typically mediated by catalytic residues that support general acid-base catalysis. This figure was made using Biorender.

Ribonucleases (RNases) are involved in numerous stages of the tRNA life cycle including biogenesis, fragmentation, surveillance, and turnover (Table 1). Nucleases targeting tRNAs are often multicomponent macromolecular assemblies that sometimes contain catalytic RNA. Protein RNases generate different cleavage ends if they are metal dependent (Fig. 1b) or independent (Fig. 1c)[6]. Another distinguishing feature of RNases is their specificity. Some RNases are tRNA-specific and rely on structural features of tRNAs, such as the elbow or anticodon stem, for recognition[7,8]. Other RNases are not tRNA specific and recognize numerous types of RNA based on other features, such as the 5′ or 3′ end. Here, we highlight how recent advances have revealed new insight into the RNases that target tRNAs, with an emphasis on human tRNA nucleases.

Table 1 (Key Resource):

Overview of human tRNA nucleases

	Nuclease	Nuclease Family	Metal Dependent	Activity	Substrate(s)
tRNA Biogenesis	nuclear RNase P (Catalytic RNA)	RNase P	Yes	endoribonuclease that removes the 5′ leader	Nuclear pre-tRNAs; additional non-coding RNA substrates
	mitochondrial RNase P(MRPP3)	PRORP/PIN	Yes	endoribonuclease that removes the 5′ leader	Mitochondrial pre-tRNAs
	RNase Z/ELAC1	RNase Z	Yes	endoribonuclease that removes the 3′trailer, repairs the 3′end	damaged tRNAs
	RNase Z/ELAC2	RNase Z	Yes	endoribonuclease that removes the 3′trailer	pre-tRNAs; additional non-coding RNA substrates
	TSEN34	TSEN/EndA	No	endoribonuclease that cleaves the 3′ splice site	Intron containing pre-tRNAs; additional non-tRNA substrates
	TSEN2	TSEN/EndA	No	endoribonuclease that cleaves the 5′ splice site	Intron containing pre-tRNAs; additional non-tRNA substrates
tRNA Fragments	RNase 1–8	RNase A	No	Large vertebrate endoribonuclease family involved in many aspects of RNA metabolism	Numerous RNAs, cleaves 3′ of pyrimidines
	Angiogenin			Stress responsive, secreted endonuclease
	RNase L	HEPN	No	Stress induced endoribonuclease activated by dsRNA and the generation of 2′−5′ oligoadenylate	Numerous RNAs, cleaves UN^N
	Dicer	RNase III	Yes	dsRNA endonuclease that generates short RNA fragments	dsRNA, pre-miRNA, tRNA with GYM motif
tRNA Surveillance and Decay	Xrn1	Xrn	Yes	Cytoplasmic 5′−3′ exoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
	Xrn2	Xrn	Yes	Nuclear 5′−3′ exoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
	Rrp6 (Exosome)	DEDD-Y	Yes	3′−5′ distributive exoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
	Dis3 (Exosome)	RNase R/II and PIN	Yes	3′−5′ processive exoribonuclease and endoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
	Dis3L1 (Exosome)	RNase R/II and PIN	Yes	3′−5′ processive exoribonuclease and endoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
	Dis3L2	RNase R/II	Yes	3′−5′ processive exoribonuclease	Numerous RNAs including pre-tRNAs and tRNA
tRNA Rescue and Defense	ANKZF1	?	No	CCA endoribonuclease	CCA end of tRNAs stalled on the ribosome
	SFLN11	SFLN	Yes	acceptor stem endoribonuclease	tRNAs
	SFLN13	SFLN	Yes	acceptor stem endoribonuclease	tRNAs
	SAMD9	ACNase	Yes	anticodon nuclease activated by viral infection	2′-O-methyl tRNA-Phe

RNases Regulate tRNA Biogenesis

tRNA maturation is a multi-step process involving two metallonucleases, RNase P and RNase Z, and two metal independent nucleases within the tRNA Splicing Endonuclease (TSEN) complex (Fig. 2a). All pre-tRNAs are transcribed with 5′ leader and 3′ trailer sequences, while only a subset of pre-tRNAs have an intron within the anticodon stem. These leaders, trailers, and introns must be removed for mature tRNA formation.

Figure 2. Nucleases of tRNA biogenesis.

(A) Schematic diagram of pre-tRNA and maturation nucleases; ribonuclease (RNase) P (green) removes the 5′ leader (light grey), RNase Z (purple) cleaves the 3′ trailer (light grey), tRNA splicing endonuclease (TSEN, blue) splices the intron (light grey) from the pre-tRNA (black). (B) Cryo-electron microscopy (cryo-EM) structure of nuclear RNase P [Protein Data Bank (PDB) ID 6AHU] [11] containing catalytic H1 RNA (green), tRNA (gold), and ten protein subunits (greys); zoom shows flipped U80 positioned to coordinate Mg (not available in structure) for attack on G1 phosphate. (C) Human mitochondrial (mt)RNase P complex (PDB ID 7ONU [12]) with the subunits colored: MRRP3 nuclease (green), MRRP1 methyltransferase (light grey), and MRRP2 dehydrogenase (dark grey). The surface of MRPP1/2 is displayed to show how MRRP1 and MRRP2 wrap the tRNA (gold) and form an extensive interface that is used to transfer it from RNase P to RNase Z to the CCA-adding enzyme. (D) Crystal structure of the long form of RNase Z from Saccharomyces cerevisiae (PDB ID 5MTZ [18]). (E) Catalytic deficient human TSEN bound to pre-tRNA (PDB ID 7UXA [26]); TSEN54 (dark grey), TSEN15 (light grey), TSEN34 (dark blue), TSEN2 (light blue). The tRNA exons are shown in gold with the part of the intron visible in the structure shown in red. Each protomer is shown in a different shade of purple. Parts of this figure were made using Biorender.

RNase P

RNase P is an endoribonuclease that removes the 5′ leader from tRNA precursors and is found across all domains of life, however its subunit composition is variable[9,10]. Humans have both nuclear and mitochondrial versions of RNase P. The human nuclear RNase P holoenzyme is a ribonucleoprotein (RNP) complex composed of ten proteins and one catalytic RNA. The structure of human RNase P revealed that the RNP forms a finger-palm-wrist clamp that holds the catalytic RNA in a conformation productive for tRNA binding and cleavage [11]. RNase P recognizes tRNA by a double anchor mechanism that involves both protein-RNA and RNA-RNA interactions. RNase P measures 12 base pairs from the tRNA TψC loop up the acceptor stem to the site of cleavage [11]. After tRNA binding, RNase P undergoes a conformational change in which U80 of the catalytic RNA moves from bulging out to, presumably, coordinate a magnesium ion (Fig. 2b).

In contrast to nuclear RNase P, the human mitochondrial RNase P (mtRNase P) lacks a catalytic RNA and is composed of three protein subunits: a nuclease (MRPP3), methyltransferase (TRMT10C/MRPP1), and dehydrogenase (SDR5C1/MRPP2). The nuclease subunit shares homology to the protein-only RNase P (PRORP) nucleases found in some bacteria and archaea[10]. PRORPs have a conserved PIN (PilT N-terminus)-like nuclease domain containing four conserved aspartate residues responsible for coordinating catalytic metal ions in the active site. The cryo-EM structure of human mtRNase P revealed that the MRPP2 tetramer along with the MRPP1 forms the tRNA recognition interface (Fig. 2c)[12]. MRPP3 relies on the MRPP1/2 subcomplex to efficiently direct it to the pre-tRNA cleavage site[13]. Interestingly the MRPP1/2 subcomplex was also shown to retain the 5′ cleaved pre-tRNA and present it to RNase Z(ELAC2) and then the CCA-adding enzyme for 3′ end processing, demonstrating coordination between different tRNA processing events[14].

RNase Z

The pre-tRNA 3′ trailer is processed by RNase Z[15]. In humans, there are two isoforms of RNase Z, often referred to as ELAC1/2 or RNase Z^S/L. ELAC1 forms a functional homodimer in a head-to-head orientation and it is believed that ELAC2 arose from an ELAC1 gene duplication event. ELAC1 localizes to the cytoplasm and does not participate in pre-tRNA processing but instead in tRNA repair after tRNA is released from stalled ribosomes[16]. In contrast, ELAC2 localizes to both the nucleus and mitochondria via alternative translational start sites, and removes the 3′ trailer of all tRNAs in both locations[17]. The structure of human ELAC2 has not been published, but the predicted Alpha Fold structure is highly homologous to the yeast crystal structure (Fig. 2d)[18,19]. Both contain two head-to-head beta-lactamase domains, fused by a linker region, with the C-terminal domain retaining the catalytic motif. This is further confirmed by a recent preprint revealing a cryo-EM structure of ELAC2 bound to mitochondrial pre-tRNA and the MRPP1/2 complex[20].

TSEN

Human pre-tRNA intron removal is catalyzed by the TSEN complex [3,21,22]. Only a subset of human tRNAs contain introns, and their purpose remains unclear[23]. The heterotetrameric TSEN complex is composed of two catalytic proteins (TSEN2/TSEN34) and two structural proteins (TSEN15/TSEN54) which appear to have evolved from the homotetrameric archaeal EndA intron splicing complex as all subunits share a fundamental core structure with EndA. The nucleases TSEN2 and TSEN34 contain the conserved catalytic triad of tyrosine, histidine, and lysine which support metal independent cleavage of the pre-tRNA at the 5′ and 3′ splice sites (Fig. 2e). The structure of the human TSEN complex was recently resolved by cryo-EM revealing how the complex recognizes pre-tRNAs with a ruler-like mechanism, coordinated in large part by TSEN54 with support from both nuclease subunits[24–27].

Little is known about the coordination of pre-tRNA processing events. It remains unclear what role additional proteins, such as the tRNA scaffold La, play in regulating tRNA maturation or if tRNA processing events happen co-transcriptionally. Recent work addressing this question suggests RNase P may help orchestrate the series of tRNA maturation events co-transcriptionally[28].

RNases Generate tRNA Fragments

A growing list of endoribonucleases have been identified that cleave tRNAs, generating different 5′ and 3′ tRNA-derived fragments (tRFs), which can regulate gene expression and translation [29–31]. tRFs are primarily generated by cleavage within the single stranded regions of TψC, D, and anticodon stem loops. Here we highlight three major classes of tRF-generating nucleases: the hRNase A family, Dicer, and RNase L (Fig. 3a).

Figure 3. tRNA fragment generating endonucleases.

(A) tRNAs are cleaved in the anticodon loop by several endoribonucleases: Angiogenin (ANG), RNase 1, RNase L. Dicer cleaves the D loop, and the TψC loop can be cut by ANG and Dicer. (B) The structure of Angiogenin (red) bound to the horseshoe shaped human placental ribonuclease inhibitor (grey) (PDBID 1A4Y [46]). The active site region is circled. (C) Structure of homodimeric RNase L with the ANK domains (greys) with 2′−5′-oligoadenylate (orange) bound, the pseudokinase domains (yellows) and the HEPN domains (pinks) which contains the active sites (circled)(PDBID 4O1P [51]). (D) dsRNA-bound (orange) Dicer in a dicing-state with two Ca²⁺ ions (magenta)(PDBID 7XW2 [61]). Parts of this figure were prepared with Biorender.

hRNase A family

The human ribonuclease A (hRNase A) superfamily comprises 13 small, secreted endoribonucleases with structural conservation to RNase A [32]. hRNases 1–8 share a canonical RNase A active site comprised of two catalytic histidine residues and a lysine. Angiogenin (RNase 5/ANG), RNase 1, and RNase 2 generate tRFs by cleaving tRNA 3′ of pyrimidines within in the anticodon loop [33,34]. Several studies suggest that other RNase A family members may also exhibit tRNA cleavage activity in vitro[32,34–36].

RNase 1 has potent tRNA cleavage activity and can target several sites within the anticodon loop of tRNA following oxidative stress[37]. RNase 1 generates numerous tRFs, including 5′ tRFs of tRNA-Gly-GCC and tRNA-Glu-CTC and 3′ halves from tRNA-Asp-GTC [37]. Its specificity for binding and cleavage is not clear. In contrast, Angiogenin is less potent than RNase 1 and appears to rely heavily on residues in the TψC loop[38]. Its active site is partially occluded by a glutamine, which may explain its less robust activity[39,40]. Nevertheless, Angiogenin plays a crucial role in tRNA cleavage during cellular stress, cleaving tRNAs primarily in the anticodon region and producing tRFs termed stress-induced tRNA fragments (tiRNAs). Although the first RNA-bound Angiogenin structure was recently solved, the structure does not show RNA directly in contact with the active site[41]. A recent preprint revealing the cryo-EM structure of Angiogenin bound to the 80S ribosome reveals how the ribosome induces rearrangements of Angiogenin’s C-terminal tail to make it much more efficient against tRNAs [42].

RNase 1 and Angiogenin are upregulated by distinct stress responses including oxidative stress (H₂O₂) and hypoxia, respectively[43]. Angiogenin is unique amongst the hRNase A family because it serves both extracellular and intracellular roles[33]. Under normal cellular conditions, Angiogenin is bound to human placental ribonuclease inhibitor (hRI) and remains inactive, but under stress is released from hRI and secreted to cleave extracellular targets, including tRNA[44]. hRI binds with femtomolar potency at the active site region (Fig. 3b)[45–47]. In the co-crystal structure of hRI and Angiogenin, hRI forms a horseshoe-like ring structure with alpha-helix-beta sheet repeats lining the outside and one end of the ring burrowing into the active site region of Angiogenin[46]. The binding of hRI to RNase 1 follows a similar mechanism[47]. RNA modifications also appear to play a role in mediating tRNA recognition and cleavage by Angiogenin. For example, Queuosine modification has been shown to interfere with Angiogenin cleavage of certain tRNAs in vitro[48].

RNase L

RNase L belongs to the metal-ion independent Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) family in which dimers assemble to create a joint nuclease active site[49]. RNase L is activated by the innate immune system. Detection of viral dsRNA leads to a signaling cascade that generates the second messenger 2′5′-oligoadentylate[50,51]. Binding of 2′5′-oligoadentylate to RNase L facilitates the homodimerization of the pseudo-kinase and C-terminal nuclease domains which twist together to form a screw-like T shape (Fig. 3c)[50,51]. RNase L shares homology with another HEPN nuclease IRE1, where one HEPN protomer binds RNA while the other protomer’s active site histidine initiates cleavage[51]. Substrate recognition by RNase L is akin to IRE1, but with slightly broader specificity, RNase L cleaves ssRNAs between nucleotides 1+ and 2+ after uridine (UN/N)[50]. This sequence recognition motif affords RNase L the ability to cleave various RNAs, including tRNA, leaving 2′,3′-cyclic phosphate and 5′-hydroxyl ends[52–54]. Importantly, like other nucleases we mention here, RNase L has a protein inhibitor, RNase L inhibitor, that obstructs 2′5′-oligoadentylate binding[55]. Additionally, small molecules have been identified that activate or inhibit RNase L[56,57]. Although the psuedo-kinase domain of RNase L is considered non-functional, small molecule inhibitors can bind to the ATP binding site and inhibit RNase L cleavage activity through disrupting dimer stability and allosterically regulating the active site[57,58].

Dicer

Dicer is multidomain L-shaped member of the RNase III family with the primary role of cleaving microRNAs and siRNAs for loading onto argonaute in the RNA-induced silencing complex[59]. Dicer leaves 5′-phosphate and 3′-hydroxyl ends, which distinguishes its products from other tRF tRNA nucleases. Two recent cryo-EM structural studies reveal the full-length structure of Dicer in apo, pre-dicing, and dicing-competent states[60,61]. In the dicing-competent state, Dicer engages dsRNA (Fig. 3d), confirming the importance of the PAZ (piwi-argonaute-zwille) and platform domains in recognizing the 3′ and 5′ ends, respectively. Dicer employs a ruler mechanism to sense its cleavage sites and cleaves RNAs approximately 22 nucleotides downstream of a 2-nucleotide overhang on the 3′ end. A human-specific helix within the PAZ domain regulates cleavage efficiently[61]. Human Dicer primarily recognizes stem loop RNAs, with a preference for short dsRNA, and was recently shown to have a sequence preference for a GYM motif (where G and the “Y” pyrimidine are paired and M is a mismatched C or A) [62]. In addition to microRNAs, Dicer can cleave certain tRNAs in the D and TψC loops[63]. The resulting tRNA fragments associate with argonaute proteins[64] and appear to drive argonaute proteins to targets through nascent RNA silencing[65]. Dicer expression and function is regulated on many levels including pre- and post-transcriptionally and through post-translational modifications[59]. Modifications to Dicer’s target RNAs, such as uridylation, can also regulate Dicer function [62].

Beyond RNase A, RNase L, and Dicer, other human RNases have been implicated in generating tRFs and much remains unknown about the generation of tRFs[1,30,31]. Given the similarities in cleavage mechanisms of RNase L and the RNase A family members, a crucial question arises: is there a shared mechanism for regulation of the fragments post-cleavage? For instance, could the 5′-hydroxyl polyribonucleotide kinase CLP1 function to regulate the turnover of the 5′-hydroxyl fragments as demonstrated for tRNA introns[22,66]; could RTCB serve to rescue cleaved tRNAs; and how do these tRNA fragments become functional to carry out their biological roles? Another interesting parallel is the strong regulation of these nucleases by inhibitors and co-factors that drive their functions. Are there other such inhibitors that exist for tRNA maturation machinery? And if so, what regulates them?

Exoribonucleases Involved in tRNA Surveillance and Decay

Given their complex secondary and tertiary structures, tRNAs are remarkably stable and resistant to decay by exoribonucleases, however loss of tRNA stability can trigger various turnover mechanisms (Fig. 4a)[1,67]. For example, the rapid tRNA decay pathway can be triggered by hypomodification of tRNAs [68]. Decay of tRNA is also highly dependent on the makeup of the 3′ end. The 3′ CCA end of tRNAs is not encoded in most eukaryotic tRNA genes and is added post-transcriptionally by the CCA-adding enzyme (TRNT1 in humans). TRNT1 plays an important role in tRNA quality control by proofreading tRNAs and marking unstable tRNAs for exonuclease decay by adding an extra CCA to the 3′ end [67,69]. Unstable tRNAs can also be targeted for decay by addition of a short poly(A) tail following the CCA[70].

Figure 4. tRNA Surveillance and Decay RNases.

(A) Following destabilization tRNAs can be targeted for destruction by Xrn nucleases and the RNA exosome through three major pathways including exposure of the 5′ end, polyadenylation of the 3′ end, or addition of an extra CCA to the 3′ end. (B) Structure of S. cerevisiae Xrn1 engaging an mRNA (orange) bound substrate on the 80S ribosome (PDBID 6Q8Y [75]). For clarity only Xrn1 and the mRNA are shown. The three terminal 5′ nucleotides are held in place by H41 and W638 and a series of conserved D residues coordinate metal ions in the active site (inset). (C) Crystal structure of the S. cerevisiae RNA exosome bound to RNA (PDBID 5VZJ [117]). Rrp6 (dark green) is located at the top of the exosome core (gray) and Dis3 (light green is located at the bottom). RNA (orange) is bound in both active sites (circled). Parts of this figure were prepared with Biorender.

Xrn Nucleases

The Xrn family of RNases are processive 5′−3′-exoribonucleases[71]. Most eukaryotes have two Xrn paralogs: Xrn2 (Rat1 in yeast) is nuclear and Xrn1 is cytoplasmic. Xrn1 and Xrn2 share two conserved N-terminal regions that form the active site core, followed by a divergent C-terminus[72–74]. Xrn nucleases rely on two major principles for RNA cleavage: ssRNA and the presence of a 5′-phosphate. The active site contains seven conserved D/E residues which coordinate two divalent metal ions for catalysis, a binding pocket for 5′ phosphorylated substrates, and histidine and tryptophan residues that sandwich the first three nucleotides of the ssRNA (Fig. 4b). A recent cryo-EM structure of Xrn1 trapped on the ribosome revealed conformational changes that support RNA binding and translocation[75], while recent NMR studies of Xrn2 revealed a highly dynamic core in which RNA binding shifts the equilibrium to the active state[76].

Stable tRNAs lack a free 5′ end and are resistant to Xrn decay. Work carried out primarily in S. cerevisiae has revealed how lack of different tRNA modifications leads to Rapid tRNA Decay (RTD) by Rat1 and Xrn1[68,77,78]. Evidence suggests the RTD pathway is conserved in humans with Xrn1 and Xrn2 likely playing critical roles in tRNA decay[1,79]. Xrn nucleases can also cleave 5′-phosphate containing tRNA fragments. For example, in yeast Xrn1 is responsible for turnover of introns following excision by the TSEN complex[80]. Although the TSEN complex generates a 5′-hydroxyl, the metazoan 5′-hydroxyl polyribonucleotide kinase CLP1 could be responsible for targeting introns to Xrns[22,66].

Given that Xrn nucleases lack substrate specificity, adaptor proteins likely play a major role in substrate recruitment and/or nuclease regulation but this has yet to be established for tRNA specific decay. Recent work has identified a family of proteins that contain a conserved Xrn2 Binding domain (XTBP) which associates with Xrn2 and regulates its activity by stabilizing the Xrn core[81–83]. Xrn activity and the RTD pathway have also been shown to be regulated by Met22 in yeast which produces a metabolite that inhibits the Xrn nucleases[1,68].

RNA Exosome & Dis3

The RNA exosome is a large multicomponent RNA processing machine, containing two nuclease subunits (Rrp6 and Dis3), that degrade numerous RNAs [84,85]. Similar to Xrn nucleases, there are nuclear and cytoplasmic forms, and the exosome cleaves both mature and precursor tRNAs[86,87]. Advances in cryo-EM have led to a number of recently determined structures of the exosome bound to a variety of different adapter proteins and RNA targets revealing the overall architecture of the complex and insight into how RNA travels through the complex[88–92]. The core of the human exosome is composed of 9 subunits (EXOSC1–9) that form a barrel through which ssRNA passes (Fig. 4c). The two catalytic nuclease subunits are positioned on opposites sides of the core.

One catalytic nuclease subunit, Rrp6 (EXOSC10), is a distributive 3′−5′ exonuclease that sits on top of the exosome core [93]. Rrp6 belongs to the DEDD-Y family characterized by a DEDD motif responsible for coordinating two metal-ions and a tyrosine that helps activate a water molecule for nucleophilic attack (Fig. 4d)[6,94]. Rrp6 contains additional domains important for RNA binding and associating with the exosome and associated factors[93]. Rrp6 associates with 3′-terminal quadruplet RNAs with a significantly higher affinity than shorter 3′-sequences[95]. This allows Rrp6 to discriminate against tRNAs with a CCA end and protects tRNAs from premature cleavage. Rrp6’s selectively for at least 4 terminal 3′ residues also provides a basis for understanding targeted decay of tRNAs after the addition of CCACCA to the 3′ end by TRNT1.

The second catalytic nuclease subunit, Dis3 (Rrp44), is a processive 3′−5′ exonuclease that sits on the bottom of the exosome core [93]. Dis3 also plays a critical role in the decay and processing of tRNA. Humans encode three Dis3 paralogs: DIS3, DIS3L1, and DIS3L2 [96]. DIS3 and DIS3L1 associate with the exosome with DIS3 being primarily nuclear while DIS3L1 is mostly cytoplasmic. In contrast, DIS3L2 functions independent of the exosome and has been linked to tRNA surveillance[97]. The three Dis3 paralogs are structurally similar to one another and belong to the larger RNase R/II nuclease family[98]. DIS3 and DIS3L1 contain an additional PIN domain with endonuclease activity. Recent cryo-EM structures of the human exosome bound to the RNA helicase Mtr4 revealed the path RNA substrates take through the barrel after unravelling by Mtr4[90]. Dis3 contains multiple interfaces that engage ssRNA, such as a conserved tyrosine residue helping clamp the RNA into position in the active site as well as several aspartic acid residues which coordinate two metal ions. In contrast to DIS3 and DIS3L1, DIS3L2 degrades structured RNAs by wedging apart dsRNA without the assistance of a helicase[98].

The exosome associates with a variety of adapter proteins and protein complexes which help with delivering RNA substrates to the exosome as well as preparing RNA substrates for decay[84]. For example, the TRAMP (Trf4/Air2/Mtr4p) complex selectively polyadenylates unmodified tRNAs, marking them for degradation by the exosome. Recent reconstitution experiments in S. cerevisiae revealed that a high level of coordination is required between TRAMP, Rrp6, and Dis3 to facilitate tRNA decay[99]. Rrp6 deadenylates the tRNA that is then passed through the exosome core and degraded by Dis3. Work in S. cerevisiae further established that the conserved lasso domain in Rrp6 binds RNA and stimulates the activity of Dis3 against hypomodified tRNAs[100]. It will be fascinating to explore if other exosome adapters also play important roles in mediating tRNA specific decay.

While the Xrn nucleases and RNA exosome have well established roles in tRNA specific decay, an outstanding question is how do these exonucleases coordinate tRNA decay. For example, CCACCA marked tRNAs are degraded by both Xrn1 and the exosome, suggesting that Xrn1 and Dis3 cooperate with one another[69]. Global profiling of mRNA decay in mouse embryonic cells revealed that Xrn1 is the major player in bulk mRNA decay while the exosome appears to play a more important role in surveillance mechanisms[101]. Given the redundancy between Xrn nucleases and the exosome for tRNA specific decay, future experiments are necessary to reveal regulation of and cell-type specific differences in tRNA decay pathways.

Quality Control and Defense RNases

In addition to the Xrn nucleases and the exosome, other RNases have recently been implicated in tRNA quality control and defense mechanisms (Fig. 5a). The nuclease ANKZF1 (ankyrin repeat and zinc finger domain-containing protein 1, Vms1 in yeast) cleaves the CCA end from peptidyl-tRNAs on stalled ribosome complexes[16,102,103]. A cryo-EM structure of a stalled ribosome bound to yeast Vms1 revealed that Vms1 binds to the 60S through multiple interfaces and makes specific interactions with the peptidyl-tRNA[104]. Vms1 contains a well conserved tyrosine residue that remodels the backbone of the tRNA to support cleavage (Fig. 5b). The cleaved tRNA, with a 2′3′-cyclic phosphate, is then recycled via a two-step process requiring removal of the cyclic phosphate by ELAC1(RNase Z) followed by addition of a new CCA end by TRNT1[16,103].

Figure 5. tRNA Defense and Rescue RNases.

(A) tRNA cartoon indicating the rough cleavage positions of ANKZF1, SLFNs, and SAMD9. (B) Cryo-EM structure of the 60S ribosome bound to VMS1/ANKZF1 from S. cerevisiae (PDBID 6R87 [104]). Vms1/ANKZf1 is a large protein that engages with the 60S subunit in several locations including adjacent to the P-site tRNA, where it utilizes Y285 to promote cleavage of the CCA end (inset). (C) Cryo-EM structure of the human SLFN11 dimer (PDBID 7ZEL [108]). The U-shaped N-terminal SLFN domain (dark green) contains the active site (circled) with a bound Mg²⁺ ion (magenta) and the tRNA acceptor stem binding interfaces. (D). Crystal structure of the SAMD9 nuclease domain bound to DNA (PDBID 7KSP [114]). Residues important for DNA/RNA binding are shown as sticks as well as putative metal ion binding residues. Parts of this figure were prepared with Biorender.

Members of the Schlafen (SLFNs) family of proteins have also been implicated in tRNA cleavage, and they support different mechanisms of translational control[105]. For example, human SLFN11 controls tRNA pools in HIV infected cells and blocks viral protein synthesis by cleaving the acceptor stem of tRNAs[106]. Recent work demonstrated that both rat SLFN13 and human SLFN11 are metal dependent nucleases that share a U-shaped N-terminal nuclease domain important for tRNA acceptor stem recognition and cleavage (Fig. 5c)[107–109]. A low-resolution cryo-EM reconstruction of human SLFN11 bound to tRNA revealed that the SLFN11 dimer binds to one tRNA, with the acceptor stem binding to SLFN through two conserved positively charged patches within the N-terminal domain [108]. An exciting area for future studies will be to determine what other SLFN family proteins function as tRNA nucleases and the basic principles that mediate different SLFN family members substrate selection.

Many microorganisms use anticodon nucleases (ACNases) as a mechanism to control translation[110,111]. For example, Kluyveromyces lactis generate Zymocin, a protein toxin ACNase which cleaves 3′ of specific modified wobble bases in yeast tRNAs, leaving a 2′,3′-cyclic phosphate and 5′-hydroxyl[112]. It was originally thought that ACNase defense mechanisms were limited to microorganisms. However recent work revealed that SAMD9 is a human ACNase that cleaves tRNA-Phe following poxvirus infection[113]. SAMD9 is a metal dependent nuclease with a preference for Mn²⁺ that has a unique specificity for tRNA-Phe with a 2′-O-methyl at the wobble position. SAMD9 cleaves the tRNA at the first phosphodiester bond within the anticodon stem. Once activated, SAMD9 effectively triggers ribotoxic stress by depleting the pool of cellular tRNA-Phe. A DNA bound structure of the SAMD9 nuclease domain revealed a series of R/K residues that support DNA/RNA binding (Fig. 5d)[114].

Concluding Remarks and Future Perspectives

The human genome encodes approximately 400 tRNA genes and beyond their essential housekeeping role decoding the genome, new roles for tRNAs are continuing to be discovered. Advances in tRNA sequencing have revealed new insights into tRNA tissue specificity, modifications, and the accumulation of different tRNA fragments and precursors[115]. RNases are key players in regulating tRNAs and their various cellular roles. Recent advances, highlighted in this review, have expanded our understanding of how different RNases recognize and process tRNA substrates, however many open questions remain (see Outstanding Questions).

Outstanding Questions Box.

• What are the different mechanisms used by cells to regulate the activity of tRNA nucleases?

• How do subcellular dynamics of both RNases and tRNAs regulate processing?

• How do different tRNA modifications impact cleavage by tRNA nucleases? What cellular cues regulate the writing and erasing of tRNA modifications?

• What role do RNA chaperones such as La play in protecting tRNAs from different cleavage events?

• Is there cooperation between different tRNA nucleases? Can some processing steps happen simultaneously or co-transcriptionally?

• Do any tRNA nucleases cooperate with other types of RNA processing machinery?

• Are there additional mechanisms of tRNA quality control mediated by tRNA nucleases that await discovery?

• What are the emerging roles for nucleases in translational control and rescue of stalled ribosomes?

• How do mutations in tRNA nucleases contribute to human diseases?

The next big breakthroughs in tRNA RNase biology are likely to come from investigating how these diverse RNases are regulated in vivo. RNases have the power to destroy a cell’s population of tRNAs, and it is vital that their activity is regulated and tuned to match the current environment of the cell. It is clear that protein co-factors and adapters are major players in regulating RNases, but much remains to be discovered about their specific roles supporting tRNA biology. In addition, tRNA modifications and the enzymes that write and erase these modifications have a significant role in controlling which tRNAs can be substrates for specific RNases. tRNAs are the most abundantly modified cellular RNAs, but aside from a few well characterized examples little is known about how the presence or absence of modifications impacts cleavage events. While many of these processes have been well characterized in yeast, the localization of many RNA processing events, such as tRNA splicing, remain unclear in humans[21]. How these regulatory events come together to accurately direct tRNA processing by nucleases underlie our basic understanding of human biology and disease as mutations in many tRNA RNases are linked to disease[3–5].

Highlights.

• Recent cryo-EM structures have offered critical new insights into the molecular mechanisms governing tRNA biogenesis.

• Structural and functional studies along with new advances in tRNA sequencing have revealed new details on the processing of tRNAs into tRNA-derived fragments (tRFs) by RNase A family members, RNase L, and Dicer.

• Recent structural advances and reconstitution experiments have provided new insights into the mechanism of surveillance and decay of tRNAs by exoribonucleases including the Xrn nucleases and the exosome.

• RNases such as the SLFN family, ANKZF1, and SAMD9 have new links with tRNA quality control and cellular defense mechanisms.

• Defining the cellular cues and molecular mechanisms that regulate tRNA processing and decay will enhance our understanding of human diseases associated with tRNA dysregulation.

Glossary

ACNase

Endoribonucleases that cleave within the anticodon loop of tRNAs

Argonaute

Family of proteins that bind small non-coding RNAs, which guide the argonautes to target RNAs

Distributive nuclease

Nuclease that only cleaves one or a few nucleotides from its substrate before release

Endoribonuclease

Nuclease that cleaves RNA from within

Exoribonuclease

Nuclease that cleaves RNA from the 3′ or 5′ end

HEPN

Higher eukaryotes and prokaryotes nucleotide binding domain. Many HEPN family members are metal ion independent nucleases which contain the RϕxxxH motif, where ϕ can be a N, H, or D residue and x is any amino acid ranging from 3 to 5 residues

PIN

Large metal dependent nuclease family characterized by the PilT N-terminal domain (PIN) which is composed of a Rossman fold

Processive nuclease

Nuclease that can continuously cleave its DNA/RNA substrate through consecutive reactions without releasing the substrate

PRORP

Protein only RNase P family that facilitate the removal of the 5′ leader sequence from pre-tRNAs

Ribonuclease (RNase)

Enzyme that hydrolyzes the phosphodiester bond of RNA

RNase R/II

Family of processive exoribonucleases that degrade RNA in the 3′ to 5′ direction

RNase III

Family of metal dependent endoribonucleases that recognize and cleave dsRNA

SLFN

Schlafen family of genes only found in vertebrates. Family members share a unique N-terminal SLFN domain

TSEN

tRNA splicing endonuclease (TSEN) complex, facilitates the removal of introns from intron containing pre-tRNAs

tRF

tRNA derived fragments