RNA

Naive T cells are poised for activation and contain a pool of translationally repressed ribosomal protein (RP) mRNA prepared to induce ribosome biogenesis to support protein synthesis, cell growth and proliferation. RP mRNA are the prototypical members of a class of transcripts initiating at cytosine followed by a CU rich element called terminal oligo pyrimidine (TOP) RNAs. TOP RNAs are regulated by an RNA binding protein LARP1, which promotes transcript stabilisation and translational repression. We investigated LARP1 function in T cell activation by generating cross-linking immunoprecipitation (CLIP) datasets detailing the LARP1-RNA interactions in naive and activated CD4+ T cells and identifying novel TOP RNAs. TOP RNAs identified by this analysis were functionally diverse. RP mRNAs were typified by high stability, and translational repression in naive T cells followed by MTORC1-dependent translation increases following T cell activation. However, other TOP RNAs varied in these aspects of their regulation. Notably, TOP RNAs with longer 3'UTRs had a relaxed dependency on LARP1 for stability and a reduced dependency on MTORC1 for their translation. Transcription start site heterogeneity also impacted TOP RNA regulation by generating a mixture of transcript isoforms with different TOP motif lengths. Longer terminal oligo pyrimidine stretches were associated with a greater dependency on MTORC1 for translation. Differential regulation of TOP RNAs may allow tuneable translational responses to MTORC1 and indicates potential roles for LARP1 beyond translation regulation and stability.

Due to its crucial role in proteobacterial RNA processing and degradation, the synthesis of the endonuclease RNase E is tightly controlled by a post-transcriptional autoregulatory mechanism governed by the 5' untranslated region of rne mRNA, the transcript that encodes it. Previous studies have identified one structural component of the rne 5' untranslated region (hp2) that helps to mediate this process in Escherichia coli by binding to RNase E. Here, we show that another component (hp3), a branched stem-loop, makes an even greater contribution to feedback regulation of RNase E synthesis and that the effects of hp2 and hp3 are additive, suggesting independent function. Despite the structural complexity of hp3, a single constituent branch appears to be the principal source of its regulatory activity. This branch also enables hp3 to bind RNase E with high affinity. The secondary structure of this branch, including the presence of two internal loops essential for its impact, is conserved in diverse Enterobacterales species, whereas the sequence of the internal loops is not. We propose that hp2 and hp3 mediate autoregulation by recruiting RNase E to the rne transcript.

Shortening of the poly(A) tail initiates mRNA decay, and the human CCR4-NOT is the principal cytoplasmic deadenylase. Although its subunits have been defined, their functions have not been comprehensively evaluated in cells. We depleted each CCR4-NOT subunit and measured transcriptome-wide effects. CCR4-NOT regulated transcripts from thousands of genes, many of which associated with the complex in cells. Distinct groups of transcripts responded according to the modular organization of the complex, while subsets exhibited unique responses to specific subunits. Poly(A) tail analysis revealed that the CNOT7 and CNOT8 deadenylases are the predominant regulators of poly(A) tail length in these cells. CCR4-NOT targets were enriched in regulatory pathways including cell proliferation, apoptosis, stress responses, and cancer-related processes. Bioinformatic analysis identified sequence determinants and candidate recruitment factors associated with CCR4-NOT regulation. The results define the breadth and specificity of CCR4-NOT-mediated mRNA decay and provide a resource for dissecting regulatory mechanisms and biological roles.

Angiogenin (ANG), a member of the RNase A superfamily, regulates multiple aspects of RNA metabolism, including rRNA processing and the generation of tRNA-derived small RNAs. However, its function during early mammalian embryogenesis remains unknown. Here, we identify a previously unrecognized requirement for ANG activity during early mouse embryonic development. Pharmacological inhibition of ANG during the first embryonic cell cycle did not prevent progression to the 2-cell stage but markedly impaired subsequent development to the 4-cell stage, indicating a critical requirement for ANG activity during the maternal-to-zygotic transition. Transcriptome-wide analysis revealed that ANG inhibition disrupted the normal remodeling of the embryonic transcriptome, characterized by persistent accumulation of a subset of transcripts that are normally reduced during the 2-cell transition, including many maternally deposited RNAs, together with altered expression of transcripts associated with zygotic genome activation. Small-RNA profiling further revealed widespread alterations in miRNA and tRNA-derived small-RNA populations. Notably, a large proportion of transcripts retained following ANG inhibition contained predicted target sites for miRNAs that were concomitantly reduced, suggesting a potential link between ANG-dependent small-RNA regulation and maternal transcript clearance. ANG inhibition was also associated with altered abundance and organization of components of RNA regulatory granules, including Tudor, TIAR, Dcp1a and Ago2, and increased association of selected retained transcripts Reep4 and Esrrb with Tudor-positive structures. Together, these findings identify ANG activity as an important regulator of RNA remodeling during the maternal-to-zygotic transition and suggest that ANG coordinates maternal transcript clearance, small-RNA dynamics and mRNP organization to support early mouse embryonic development.

N6-methyladenosine (m6A) RNA modifications regulate diverse cellular processes, including the response to virus infection. However, how m6A controls antiviral gene expression and whether effects on viral replication are due to direct modification of viral RNA or regulation of cellular transcripts remain unclear. Herein, we analysed how m6A modifications control the cell-autonomous innate immune response to influenza A virus (IAV) infection. Depletion or inhibition of the m6A writer METTL3 or the m6A reader YTHDF2 restricts IAV replication. However, using a quantitative method to identify m6A sites, we did not detect substantial m6A modification of viral RNA. In contrast, many IAV-induced cellular transcripts were m6A modified in their coding region, including those that encode pattern recognition receptors and direct antiviral proteins. Inhibition of interferon signalling restored IAV replication after METTL3 inhibition, highlighting that m6A controls the antiviral response to infection. METTL3 inhibition increased expression of the paralogous antiviral proteins IFITM2 and IFITM3, which were required for the antiviral activity of the METTL3 inhibitor. We propose that m6A modifications regulate the expression of antiviral proteins to limit the magnitude of their induction and balance antiviral activity with maintaining cellular homeostasis.

Noncoding RNAs commonly rely on RNA-binding proteins (RBPs) to execute their regulatory functions, but whether RBPs can instead facilitate target recognition by noncoding RNAs is poorly understood. Here, we identify a non-canonical function for Pumilio proteins PUM1 and PUM2 in gene regulation as cofactors of the viral small nuclear RNAs HSUR1 and HSUR2 encoded by Herpesvirus saimiri. HSUR1 and HSUR2 function as microRNA and RBP adaptors that bind target mRNAs to downregulate their expression. We show that HSUR1 and HSUR2 contain conserved Pumilio response elements (PREs) through which they associate with PUM1 and PUM2, without measurably altering PUM protein activity or subcellular distribution in infected cells. Instead, PUM binding is required for HSUR-mediated mRNA repression. Mutation of the PREs abolishes PUM association and HSUR-mediated repression without disrupting HSUR association with microRNAs, whereas tethering PUM1 or PUM2 directly to PRE-deficient HSURs restores repression. Furthermore, HSURs unable to bind PUMs fail to associate with target mRNAs, demonstrating that PUM binding is required for productive HSUR-mRNA engagement. These findings reveal that PUM proteins can function as enabling factors for noncoding RNA target recognition rather than as downstream effectors of repression, expanding the functions of these RBPs in RNA-mediated gene regulation.

Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [≥] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [≥]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [≥]50%, coverage [≥]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.

Circular RNAs (circRNAs) are covalently closed transcripts that lack a 5' cap, consequently their translation depends on cap-independent initiation, in most cases through Internal Ribosome Entry Sites (IRESs). Whereas IRES-mediated translation has been studied almost exclusively in cis, the use of IRES-containing circRNAs to activate translation of an independent mRNA in trans has only recently begun to be explored. Here, we characterize SynFarsa, a synthetic circRNA-derived IRES-like element that promotes cap-lindependent translation in mouse and human cells. We engineered SynFarsa into a trans-acting regulator by fusing it to a short antisense sequence complementary to a selected region of a target mRNA. This reproduces the architecture of a circular SINEUP, in which IRES activity promotes translation in trans of a transcript recognized through antisense complementarity. Directing this construct to the hsp90 3'UTR increased HSP90 protein levels approximately 1.6-lfold without altering hsp90 mRNA abundance, whereas the hsp70 3'UTR and the 5'UTRs of both transcripts were not permissive, indicating that activity depends strongly on target site selection. In a FUSP525L neuroblastoma model of Amyotrophic Lateral Sclerosis, this increase in HSP90 was accompanied by a reduction in the number and size of cytoplasmic FUS aggregates. SynFarsa thus provides a circRNA-derived effector domain for targeted translational upregulation of an endogenous protein.

The self-splicing group I intron ribozyme from Tetrahymena thermophillia (THr) is a prototypical example of how RNA overcomes its limited chemical repertoire to form complex, negatively charged junctions and catalyze chemical reactions. Despite over 30 years of biochemical and structural characterization, questions still remain regarding the requirements for THr catalysis that may inform structure based drug design against group I introns, as well as the use of THr as an RNA editing tool. Recent cryo-EM structures have provided an unprecedented window into the first and second steps of splicing, enabling detailed mechanistic investigation at the atomistic level. In this work, we leverage solvation theory, molecular dynamics, alchemical free energy simulations, and machine learning accelerated QM/MM to probe outstanding questions related to 1) the role of a possible third Mg2+ binding in the active site, 2) differences in catalytic requirements between Gexo cofactors and between the first and second steps of splicing, 3) the potential role of an acid or base, and 4) restrictions on the identity of the 5' exon splice site nucleotide, U(-1). We propose that a transient, cofactor dependent, interaction with a third metal participates in the first step, while a higher affinity interaction with this third ion is required in the second step. We also propose that the presence of a phosphate on the Gexo in the first step, and analogously the G{omega} in the second step, forms a metal ion binding site not present when guanosine is bound. Finally, we propose and computationally test design modifications of G22 (isofunctional substitutions of 2,6-diaminopurine that tune the pKa at the N1 position) that we predict will enable the accommodation of C(-1) in a crucial wobble-like pair at physiological pH. This design would provide a potential strategy for expanding the sequence space accessible to programmable trans-splicing ribozymes in RNA editing applications.

Double-stranded RNAs are inflammatory byproducts of in vitro transcription of single-stranded RNA. Here we investigate removal of dsRNA byproducts using dsRNA affinity chromatography (dsRNA-AC) and two common mRNA molecules: 1) GFP, and 2) high dsRNA wild-type firefly luciferase (fLucWT). For GFP mRNA, dsRNA levels decreased by 2,000-fold, and mRNA recovery was high. In contrast, dsRNA levels decreased only two-fold for fLucWT, and mRNA yields were lower. Biophysical characterization revealed that fLucWT contains polydisperse higher-order structures that interfered with dsRNA-AC. Sedimentation-velocity analytical ultracentrifugation experiments demonstrated the higher-order structures were driven by mRNA concentration. Decreasing fLucWT concentrations increased the effectiveness of dsRNA-AC by reducing polydispersity. dsRNA levels were measured with ELISA, dot-blots, immuno-northern blots, and a cell-based interferon release assay, enabling comparison and cross-validation of these analytical methods. dsRNA-AC is a new chromatography modality, and this study identifies sample polydispersity as a key determinant of efficient dsRNA removal.

Transfer RNA (tRNA) modifications regulate protein synthesis, yet their sequence positions and stoichiometries are known only for a subset of tRNAs in model organisms. Aplysia californica is a key neurobiological model in which neuronal modified ribonucleosides have been linked to changes in animal behavior. However, Aplysia tRNA modifications have not been mapped, obscuring their roles in modulating neuronal functions. To address this knowledge gap, we leveraged hybridization pulldowns and an integrated mass spectrometry (MS) strategy combining nucleoside profiling, intact tRNA measurements, and bottom-up tRNA modification mapping to determine the full sequences of Aplysia tRNAGluCUC and tRNALysUUU. Sub-stoichiometric modifications including dihydrouridine (D), 5-methylcytidine (m5C), 5,2'-O-dimethyluridine (m5Um) and 1-methyladenosine (m1A) on tRNAGluCUC varied across tissues, suggesting tRNA modifications are regulated to accommodate translational needs. In addition to conserved thio-modifications in the anticodon loop of tRNALysUUU, we report 2-thiocytidine (s2C) at position 32, representing the first detection of s2C in eukaryotes. We also developed a generalizable method to directly detect tRNA halves by intact LC-MS, revealing endogenous tRNA cleavage patterns. Our findings illustrate the advantages of combining intact and bottom-up MS, reveal s2C as an unexpected tRNA modification in a classical neurobiological model and offer new insights into post-transcriptional regulation of animal physiology.

TDP-43 aggregation is a pathological feature of several neurodegenerative diseases, resulting in nuclear loss of function and RNA dysregulation, including cryptic exon derepression. TDP-43 comprises two RNA recognition motifs required for canonical RNA binding, a low-complexity domain implicated in phase separation, and an N-terminal domain (NTD) that mediates oligomerization. While NTD-driven oligomerization is critical for TDP-43 splicing function, some studies suggest a direct interaction of NTD with RNA. Here, we investigated RNA binding by the NTD and its functional interplay with oligomerization. We show that the NTD preferentially binds UG-rich RNA via residues located at the oligomerization interface. The NTD engaged RNA only on monomeric TDP-43, as RNA binding and oligomerization compete for the same interface and rely on similar interaction modes. By disentangling these competing activities, we demonstrated that oligomerization, but not NTD-RNA interaction, is essential for TDP-43-dependent alternative splicing and repression of cryptic exons. Our findings uncover a previously unappreciated RNA-binding activity within the TDP-43 NTD that directly competes with oligomerization and establish oligomerization as a key determinant of TDP-43 cryptic exon repression.

Background: X-chromosome inactivation (XCI) evolved independently in eutherian and marsupial mammals, where it is orchestrated by the unrelated long non-coding RNAs Xist and RSX, respectively. Xist organizes a repressive nuclear compartment through multivalent RNA-protein interactions, but whether RSX exploits similar biophysical principles remains unknown. A recent paper has identified bona fide RSX interacting proteins. Results: We integrated proteome-scale RNA-protein interaction prediction, experimental validation, phase-separation propensity analysis, functional annotation and comparative RNA-structure modelling to characterize the RSX interaction landscape. Using catRAPID, we ranked 1,168 RNA-binding proteins from the native Monodelphis domestica proteome. Predictions were significantly enriched for experimentally identified RSX interactors, with 4.85-fold enrichment among the top 50 candidates (P approximately 1.6 x 10-5), increasing to approximately eightfold for proteins shared by the experimental RSX and Xist interactomes (P approximately 2 x 10-6). Among 30 high-confidence RSX interactors, 13 were experimentally supported, 17 were previously unrecognized candidates and 17 exhibited high phase-separation propensity. The network was enriched in ribonucleoprotein granules and nuclear bodies and converged on m6A regulators and SR-family splicing factors. Comparative modelling detected no conserved secondary or tertiary architecture between RSX and Xist. Conclusions: RSX and Xist appear to have converged not through RNA sequence or global structure, but through recruitment of related, condensation-prone protein networks. These findings identify interaction-network and biophysical convergence as a potential principle of lncRNA-mediated chromosome regulation and provide testable candidates for determining whether RSX establishes a condensate-like compartment on the marsupial inactive X.

RNA structure governs the function of non-coding RNAs and influences mRNA stability and translation, making testing of models of experimental RNA structure relevant to fields spanning structural biology, virology, and molecular therapeutics. Here we present FAST-MaP (Fast and Accessible Sequencing Technology for Mutational Profiling), a protocol that enables per-nucleotide RNA structure characterization using standard molecular biology equipment and requiring no sequencing infrastructure or bioinformatics expertise. RNA is chemically modified with orthogonal probes (2A3 and DMS), reverse-transcribed, and PCR-amplified to produce dsDNA amplicons that are submitted directly to a commercial primer-less sequencing service. Returned FASTQ files are processed through a freely accessible web server to generate normalized reactivity profiles within minutes. The complete protocol, from DNA template to structural data, can be completed in approximately one week. We illustrate the workflow on a 659-nucleotide RNA, demonstrating how to test structure preservation across buffers, and how to test specific secondary and tertiary structure predictions of the RNA from computational modeling or cryo-electron microscopy. The protocol requires only standard molecular-biology skills and does not require sequencing or bioinformatics expertise.

The 5'-untranslated region (5'-UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly structured and comprises five conserved stem-loops that serve as cis-acting regulatory elements. During infection, host proteins are recruited to these elements and regulate multiple steps during the viral life cycle. Stem-loop 5 (SL5) is highly conserved among different alpha- and betacoronaviruses and includes the start codon of non-structural protein 1 (nsp1). However, the function of SL5 in viral replication and its interacting proteins remain to be determined. Here, we identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a host protein functionally interacting with SL5. HNRNPM translocates into the cytoplasm during SARS-CoV-2 infection and promotes protein synthesis from the viral 5'-UTR. Mutations in the lower stem of SL5 impair HNRNPM binding and reduce protein synthesis. Notably, the interaction between HNRNPM and SL5 is conserved among different alpha- and betacoronaviruses. Our findings indicate that HNRNPM is an important host factor that regulates viral translation to promote SARS-CoV-2 replication.

The long-range RNA-RNA interaction between the termini of a flavivirus genome is controlled by multiple local RNA secondary and tertiary structures. However, a complete picture of this essential genome cyclization process is still elusive. Herein, by using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), SHAPE and mutational profiling, in vitro RNA binding assays, and reverse genetics approaches, we demonstrated that the hairpin 2 in the capsid-coding region (cHP2) is required for efficient replication of yellow fever virus and functionally interacts with the 3'-small hairpin (sHP) during genome cyclization. Interestingly, through a combination of multidimensional chemical probing and functional characterization assays, it was discovered that the sHP forms previously unknown tertiary interactions with an immediately upstream GACG region embedded within the 3'-cyclization sequence. This crucial tertiary RNA motif accounts for a potential negative regulatory mechanism of genome cyclization and is universally conserved among the mosquito-borne-related flaviviruses, which constitute a major phylogenetic branch within the subgenus Euflavivirus, genus Orthoflavivirus. Our study highlights the intricacy of RNA-based regulation in flaviviruses.

Trypanosomatids are medically important single-celled eukaryotic protists with exceptional RNA biology. They display highly unusual RNA biogenesis pathways, notably mitochondrial RNA editing, an essential post-transcriptional process transforming cryptic primary transcripts into functional mRNAs. Large multiprotein assemblies coordinate the RNA editing process, in which small guide RNAs (gRNAs) anneal to the pre-mRNAs and serve as templates for uridine insertion or deletion. Kinetoplast RNA Editing Helicases (KREHs) are important components of the RNA editing process that modifies large portions of pre-mRNA via the sequential annealing and dissociation of multiple gRNAs. However, how KREH helicases interact with components of the editing machinery, and which subunits mediate these interactions, remain unresolved. Here, we examined an association between the DEAH/RHA RNA helicase KREH2 and the RESC1-RESC2 protein heterodimer using proximity labeling and co-immunoprecipitation coupled to quantitative mass spectrometry in vivo. We reconstituted a minimal trimeric KREH2 peptide-RESC1-RESC2 complex in vitro and characterized novel direct binding of a KREH2 peptide to RESC1 using cryo-electron microscopy. These results establish a mechanistic link between the RNA helicase KREH2 and the RESC1-RESC2 module at the 5'-end of gRNAs, providing new avenues for studying the orchestration of RNA remodeling in trypanosomatid mitochondrial RNA editing.

Mitochondrial pre-mRNA editing in kinetoplastids, a clade that includes human-infective parasitic protists such as Trypanosoma and Leishmania, is required to produce translatable, functional gene products from otherwise nonfunctional mitochondrial precursor transcripts of essential respiratory chain complex subunits. RNA editing is a template-guided process in which cognate guide RNAs (gRNAs) anneal to specific sites at the pre-mRNA and direct the complementary insertion and/or deletion of uridines. Large multi-modular complexes - like the RNA-Editing Substrate-binding Complex (RESC) and the RNA-Editing Catalytic Complexes (RECC) - organize and catalyze RNA editing, respectively. Within these assemblies, Kinetoplast RNA Editing Helicases (KREHs) play an important role in ribonucleoprotein complex remodeling, particularly in pan-editing, when transcripts are extensively modified throughout their length by multiple gRNAs. However, the mechanisms by which KREH RNA helicases facilitate pan-editing, and their specific roles in this process, remain unclear. Here, we define the molecular properties of the Trypanosoma brucei DEAD-box RNA editing helicase KREH1. Cellular co-proteome analyses reveal that KREH1 interacts with subunits of the RESC complex and biochemical experiments show that KREH1 is an RNA-dependent ATPase that unwinds double-stranded RNA substrates. We show that the flexible C-terminus of KREH1 is important for RNA binding and, consequently, for enzymatic activity in vitro and interactions with the RESC complex in vivo. Overall, our experiments refine the role of KREH1 in the pan-editing process and establish its utility as a molecular tool for investigating higher-order RNA editing complexes in the cellular context.

Nanopore direct RNA sequencing has emerged as a powerful technology for studying RNA and modifications at single-molecule resolution. Although originally developed for polyadenylated RNAs, protocol adaptations have extended its application to non-polyadenylated RNA species, including tRNAs and rRNAs. However, the lack of open and flexible (de)multiplexing solutions remains a limitation for large-scale sequencing studies. Existing community-driven approaches enable multiplexing of polyadenylated RNAs and, more recently, tRNAs, but do not fully address the challenges of targeted sequence-specific Nanopore sequencing of non-polyadenylated RNAs. Here, we present an improved sequence-specific direct RNA sequencing protocol for ribosomal RNAs, achieving enhanced capture and longer read lengths as demonstrated for human 28S rRNA. We complement this protocol with an expanded WarpDemuX software toolbox for flexible sequence-specific (de)multiplexing. We validate the approach using human cell lines carrying knockouts of different C/D box snoRNAs involved in site-specific 2'-O-ribose methylation of 28S rRNA. Our results demonstrate efficient multiplexed detection of sequence-specific RNA molecules and associated modification changes. While established here using rRNA, we validate our approach on another panel of non-polyadenylated RNA species, providing an open and scalable framework for targeted direct RNA sequencing and RNA modification analysis

Ribonuclease Z enzymes remove 3' trailers from precursor tRNAs. In human cells, ELAC2/RNase ZL matures nuclear and mitochondrial pre-tRNAs, whereas the cytosolic short paralog ELAC1/RNase ZS repairs ANKZF1-cleaved tRNAs, but its broader substrate repertoire remains unclear. We investigated ELAC1 function using transcriptome profiling, small RNA sequencing, CLIP-seq, and biochemical reconstitution. ELAC1 was dispensable for canonical tRNA processing but associated with structured RNA polymerase III transcripts, prominently Y RNAs and vault RNAs (vtRNAs). Purified ELAC1 cleaved all four human vtRNAs near their 3' ends, with high-resolution mapping identifying a conserved cleavage position within the 3' extension. Efficient cleavage required structural features of the vtRNA terminal region. In contrast, ELAC2 showed little or no vtRNA cleavage under the conditions tested and displayed stricter substrate and cleavage-site selection on pre-tRNA. Loss and re-expression of ELAC1 altered structured ncRNA abundance and small-RNA end profiles. These findings identify vtRNAs as direct ELAC1 substrates and support functional specialization of human ELAC paralogs, with ELAC2 favoring precise pre-tRNA maturation and ELAC1 exhibiting broader RNA substrate specificity.