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.

Small RNAs (sRNAs) regulate the expression of many genes including those involved in antibiotic resistance and bacterial virulence, making them potential therapeutic targets. A molecule that binds an sRNA could interfere with its ability to bind its target mRNA and disrupt the regulation mechanism. Randomization and screening of natural arginine rich motif (ARM) peptides led to peptides capable of interfering with the sRNA MicF's ability to regulate ompF in Escherichia coli. Molecular dynamics simulations suggested that this effect was not a result of a direct disruption of the MicF-ompF interaction. Instead, the peptides interfere with binding of the chaperone Hfq, which is required for MicF-mediated regulation. Subsequent testing demonstrated peptide specificity for MicF over two other Hfq scaffolds and the ability to disrupt regulation of two additional MicF targets. Together, these findings support the use of synthetic ARMs as a potential tool for modulating sRNA function in bacteria.

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.

Filoviruses, such as Ebola virus (EBOV), are highly pathogenic non-segmented negative-sense RNA viruses (nsNSVs) with limited therapeutic options. Filovirus RNA structures remain largely untapped due to the enhanced biosafety requirements for handling infectious virus. Here, we present the first in-cell secondary structure maps of four EBOV mRNAs (VP35, VP40, VP30, and VP24) using two orthogonal chemical probing approaches: SHAPE-MaP and fbDMS-MaP. We find that EBOV mRNA coding sequences (CDS) are highly structured, much like +ssRNA viruses, whereas untranslated regions (UTRs) are significantly less structured. This suggests that high CDS structure contents are general features of viral translation templates, and that nsNSVs have evolved separate regulatory function at the RNA structure level that extends beyond using distinct mRNAs and genomes. These structure maps are consistent with formation of mRNA 5' hairpin structures during infection and reveal numerous additional RNA structures within the CDS, 3' UTRs, and at CDS-UTR junctions. To assess functionality, we disrupted these structures with locked nucleic acid (LNA) antisense oligonucleotides. Disrupting the TSS hairpins in VP35, VP30, and VP24 decreased infection by >60%, indicating these mRNA structures are critical for infection. LNA targeting of the newly identified structures reduced EBOV infection by 31% to 88%, thereby linking RNA structural integrity to viral function. Synonymous mutation rates and covariation analysis provided evolutionary support across mammalian filoviruses for the functional RNA elements observed. Collectively, these results demonstrate EBOV mRNAs contain numerous conserved RNA motifs contributing to viral infection, and that these elements represent promising targets for development of pan-filoviral therapeutics.

Satellite RNAs (satRNAs) are parasitic subviral agents whose biological activities are mediated largely by specific sequence determinants and structured RNA elements. Establishing biologically supported global RNA structures is therefore essential for understanding how RNA architecture underlies satRNA functions. Cucumber mosaic virus (CMV) satRNA is one of the best-characterized models for investigating satRNA structure-function relationships; however, a biologically supported global RNA architecture of CMV satRNA has yet to be established. Here, we applied AlphaFold3 modeling to predict the global structure of CMV satRNA T1 (sat-T1). Initial full-length structure modeling generated multiple long-distance interactions that lacked biological support. We therefore used fragment-based modeling, combined with chemical probing, evolutionary covariation, and compensatory mutagenesis, to derive a biologically supported global secondary structure. To determine whether structurally distant regions could interact in the context of the full-length RNA, we engineered a structure-guided T1-ZD mutant that preserved the supported secondary structure while reducing alternative base-pairing possibilities. Full-length AlphaFold3 modeling of T1-ZD largely recapitulated the proposed architecture, while one predicted model revealed a long-distance interaction that was subsequently supported by compensatory mutagenesis analysis. These findings suggest that the 3' terminus of sat-T1 may undergo conformational switching between alternative structural states. Together, our work establishes a biologically supported global RNA architecture for CMV sat-T1 and provides a structural framework for investigating the molecular basis of satRNA function.

RNAs regulate gene expression and cellular processes, often relying on specific conformations for function. RNA folding is hierarchical and sequence-dependent, with nearest-neighbor thermodynamic models commonly used to predict secondary structure. Current models were developed using optical melting experiments in 1 M NaCl, which does not represent the cellular environment. To address this, we developed a new model in Advanced Dulbecco's Modified Eagle Medium (Adv. DMEM), which mimics mammalian extracellular ionic composition. This in vivo-like model provides RNA folding parameters for helical base stacks and loop motifs. Optical melting experiments revealed helical stacks, particularly tandem G-U pairs, are less stabilizing in Adv. DMEM. Loop parameters were generally destabilizing but highly dependent on both sequence and loop type, with internal loops displaying idiosyncratic behavior. Structure prediction benchmarking revealed minimal differences overall, except for tRNAs, which showed improved prediction reliability and enhanced cloverleaf stability. Notably, tRNAs lack internal loops, suggesting further studies in Adv. DMEM could refine secondary structure predictions. This in vivo-like parameter set is included in the RNAstructure software package. Grounding these parameters in a physiologically relevant environment, we improve the biological relevance of RNA secondary structure predictions and establish a foundation for studying RNA folding under in vivo conditions.

Intrinsically disordered regions are widespread in RNA-processing machines. In Escherichia coli, RNase E uses its intrinsically disordered C-terminal domain (CTD) to recruit RNAs to the N-terminal catalytic domain, including mRNAs targeted by regulatory small RNAs (sRNAs), but the basis of substrate recognition and specificity is unclear. We engineered a protease-cleavable RNase E and used split-CRAC to isolate RNAs crosslinked to the AR2 sub-domain of the intrinsically disordered CTD fragment. AR2 preferentially engaged mRNAs and was depleted of sRNAs and sRNA-containing hybrids, supporting recognition of the mRNA. AR2 contacts concentrated on accessible A-rich motifs surrounding ribosome-binding sites and start codons, and purified AR2 recognised this motif in vitro. AR2 also contacted an AUAA motif in the rne translation-initiation region, and AR2 deletion increased RNase E abundance implicating this interaction in autoregulation. These findings define a relatively short AR2-binding motif and are consistent with CTD interactions with the 30S subunit that may provide additional specificity for a subset of mRNA translation initiation regions. SIGNIFICANCE STATEMENTMost RNA turnover in bacterial cells is carried out by the RNA degradosome, yet how this molecular machine checks and selects RNAs for degradation remains incompletely understood. We show that an intrinsically disordered region of the degradosome enzyme RNase E, termed AR2, preferentially binds A-rich sequences near sites of translation initiation. Through recognition of this shared sequence feature in a common functional context, AR2 may help the degradosome recognise messenger RNAs as a functional class. AR2 also contributes to feedback control of RNase E expression by recognising its own messenger RNA.

RNA granules are biomolecular condensates that are reversibly formed through multivalent interactions among mRNAs and RNA-binding proteins (RBPs). RNA granules include stress granules and neuronal transport granules, which share common components and functions in translational regulation. Fragile X messenger ribonucleoprotein (FMRP) is an RBP encoded by FMR1, whose loss of function causes fragile X syndrome (FXS), a neurodevelopmental disorder. FMRP is a component of both stress granules and neuronal transport granules. Although several FXS-associated missense mutations in FMRP have been identified, it remains unclear whether these mutations affect the assembly of FMRP into RNA granules. Here, we found that the KH1 domain mutation G266E markedly reduced the accumulation of FMRP in stress granules in HEK293T cells, whereas the KH2 domain mutation I304N had little effect. In addition, both G266E and I304N reduced the accumulation of FMRP within neuronal transport granules in FMRP-deficient mouse cortical neurons. Because G266E and I304N have been reported to impair the RNA-binding and/or RNA-associated functions of FMRP, our findings suggest that the KH1 and KH2 domains are important for the assembly of FMRP into RNA granules. These findings further raise the possibility that impaired RNA granule assembly may contribute to the pathogenesis of FXS and other neurodevelopmental disorders.

Background: RNA molecules form thermodynamic ensembles, but interpretation often requires a single representative structure. Existing base-pair centroid estimators assess agreement at the level of individual base pairs and do not directly target nesting depth along the sequence. Methods: We introduce Mountain Centroid, which minimizes expected squared mountain-profile distance, and derive dynamic programming algorithms with and without RNA pairing constraints. We also combine the Mountain Centroid objective with the base-pair centroid gain. Results: Across 21,254 RNAStrAlign sequences, Mountain Centroid had lower median normalized mean squared mountain distance (NMSMD) than minimum-free-energy (MFE) and base-pair centroid ({gamma} = 1) structures, whereas its median base-pair F1 was lower. Imposing RNA pairing constraints improved base-pair F1 for 59.35% of sequences and reduced it for 3.58%. At an illustrative weight, the combined objective had median base-pair F1 similar to MFE while retaining lower median NMSMD than MFE and all tested {gamma}-centroid settings. Conclusions: Mountain Centroid represents an RNA structural ensemble with a single secondary structure that reflects how nesting depth varies across nucleotide positions. Combining mountain-profile and individual-base-pair criteria allows their relative contributions to be varied.

The RNA exosome is an essential, evolutionarily conserved ribonuclease complex that processes and degrades many classes of RNA. The complex is composed of three structural cap subunits (EXOSC1-3/Csl4, Rrp4, Rrp40; H. sapiens/S. cerevisiae), six structural core subunits (EXOSC4-9/Rrp41,Rrp46,Mtr3,Rrp42,Rrp43,Rrp45), and a catalytic ribonuclease (DIS3 or DIS3L/Dis3). Cofactors that associate with the RNA exosome confer specificity to target specific RNAs for processing and/or decay. Missense mutations in genes encoding structural subunits of the RNA exosome have been linked to neurological diseases. Notably, several pathogenic mutations have been identified in EXOSC3 that are associated with pontocerebellar hypoplasia type 1b (PCH1b). These pathogenic alleles cause a broad spectrum of clinical severity, suggesting variant-specific functional consequences. Given the high degree of conservation between the human and budding yeast RNA exosome complexes, we performed a systematic analysis of eight pathogenic EXOSC3 variants modeled in budding yeast Rrp40. We find that two Rrp40 variants cause growth defects, show distinct negative genetic interactions with RNA exosome cofactor mutants, and impair RNA processing in budding yeast. One of these variants, EXOSC3-Y109N/Rrp40-Y64N, had not been previously characterized in any mechanistic studies. Computational stability predictions and immunoblot analyses indicate that most EXOSC3/Rrp40 variants display reduced steady-state protein levels, but decreased protein levels do not strictly correlate with phenotype or disease severity, suggesting that individual variants disrupt RNA exosome function through distinct mechanisms. Collectively, our studies suggest that pathogenic EXOSC3 variants alter RNA exosome function through distinct mechanisms and provide insight into the specific molecular defects that could underlie pathology.

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.

Previous studies revealed a crosstalk between intronic polyadenylation (IPA) and the DNA damage response (DDR). Indeed, genotoxic agents, including radiations and anticancer drugs (e.g., cisplatin that crosslinks DNA), regulate the ratio of IPA to last-exon transcripts in many genes. Conversely, multiple genes involved in the DDR, especially homologous recombination, are regulated at the IPA level. The U1 small nuclear RNA (snRNA) widely represses IPA, thereby enhancing full-length gene transcription. However, besides its implication in IPA regulation by ultraviolet-C radiation, little is known about U1 snRNA effects on the DDR and on cell sensitivity to genotoxic agents. Here, we show that U1 snRNA blockade using an antisense oligonucleotide (U1-AMO) in lung cancer cell lines enhances cell growth inhibition by cisplatin, through an increase in cisplatin-induced DNA damage. 3-seq analysis indicates that U1 snRNA blockade represses full-length mRNA expression of multiple genes of the nucleotide-excision repair and Fanconi anemia pathways, which are involved in the repair of cisplatin-DNA crosslinks. Our 3-seq analyses also reveal that moderate doses of U1-AMO and cisplatin upregulate the IPA:LE isoform ratio in overlapping but distinct sets of genes, and that U1-AMO prevents cisplatin effects on the IPA:LE ratio in a large subset of genes. Altogether, these data extend the crosstalk between IPA and the DDR and suggest that U1 snRNA targeting may be used to sensitize cancer cells to genotoxic agents.

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.

RNA recognition motif (RRM) proteins frequently contain multiple RNA-binding domains connected by flexible linkers, yet the contribution of transient interdomain interactions to RNA recognition remains incompletely understood. Here, we investigated the structural organization of the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl) using solution NMR spectroscopy in combination with rational protein engineering, restrained docking and RNA-binding studies. Progressive extension of the native interdomain linker resulted in a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, demonstrating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as two independently tumbling domains. NMR-guided docking identified a compact arrangement compatible with the experimental data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface produced the opposite effect: instead of increasing interdomain mobility, it exhibited enhanced rotational coupling while remaining natively folded, indicating a redistribution of the conformational ensemble rather than disruption of the domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant additionally diminished sequence discrimination, demonstrating that perturbations shifting the conformational equilibrium in either direction compromise RNA recognition. Together, our results demonstrate that RNA recognition by Sxl is governed not by a single apo structure but by a finely balanced conformational ensemble, and that perturbing this equilibrium in either direction compromises high-affinity and sequence-selective RNA binding.

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.

Accurate annotation of RNA base-pairing interactions is essential for structural analysis, benchmarking, and data-driven RNA structure prediction. Several tools can extract RNA interactions from three-dimensional coordinates, but their outputs are heterogeneous and may disagree, particularly for non-canonical base pairs. We present EXTRARNAS, a Java-based framework for automated, reproducible, and user-friendly large-scale extraction of RNA structural annotations with multiple tools. EXTRARNAS processes batches of RNA structures specified by PDB identifier and chain, or provided as local PDB files, executes annotation tools through a Docker-based environment, and parses tool-specific outputs using ANTLR4-based grammars. For each structure-tool pair, the framework generates standard BPSEQ files for canonical cis Watson-Crick interactions and introduces BPSEQE, a standardized text format for representing the extended secondary structure, preserving canonical, non-canonical, and multiple interactions per nucleotide. The current prototype supports RNAView, MC-Annotate, and RNAPolis Annotator. We demonstrate EXTRARNAS on eight RNA structures containing triple-helix motifs, comparing extracted canonical pairs against curated BPSEQ references and evaluating the recovery of manually validated Hoogsteen interactions. The results show consistent differences among tools, especially for non-canonical interactions, highlighting the need for standardized representations such as BPSEQE to support reproducible comparison and future consensus-based annotation.

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 recent discovery of obelisks, novel deltaviruses, and other viroid-like circular RNAs has greatly expanded the known diversity of autonomous RNA elements. However, cold terrestrial ecosystems remain comparatively undersampled for these molecules. Using a structure-based modification of the Tormentor pipeline, we assembled two putative non-coding rod-like circular RNAs from winter metatranscriptomes of oroarctic tundra soil collected near Kilpisjarvi, Finland. Sequence-based searches identified no meaningful homologs for either RNA. Structure-based analyses identified affinities to deltavirus-like ribozymes in Rod 1 and to hammerhead ribozymes in Rod 2, with structural compatibility confirmed by integration into an established covariation model. Rod 1 was additionally detected in independent alpine metatranscriptomic datasets, whereas Rod 2 was not identified beyond the original sample. Collectively, these results identify Rod 1 as a strong candidate viroid-like RNA bearing a putative ribozyme-like structure motif that warrants experimental validation, while Rod 1 remains a preliminary candidate requiring further confirmation.