Competition between two HUSH complexes orchestrates the immune response to retroelement invasion.

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

Endogenous retroelement expression in the gut microenvironment of people living with HIV-1.

BACKGROUND: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). METHODS: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. FINDINGS: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. INTERPRETATION: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. FUNDING: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).

T-cell immunity against senescence: potential role and perspectives.

The development of age-associated diseases is related to the accumulation of senescent cells in the body. These are old non-functional cells with impaired metabolism, which are unable to divide. Such cells are also resistant to programmed cell death and prone to spontaneous production of some inflammatory factors. The accumulation of senescent cells is related to the age-associated dysfunction of organs and tissues as well as chronic inflammation that enhances with age. In the young organism, senescent cells are removed with the innate immunity system. However, the efficiency of this process decreases with age. Nowadays, more and more evidences are accumulating to support the involvement of specific immunity and T-lymphocytes in the fight against senescent cells. It has great physiological importance since the efficient elimination of senescent cells requires a high diversity of antigen-recognizing receptors to cover the entire spectrum of senescent-associated antigens with high precision and specificity. Developing the approaches of T-cell immunity stimulation to generate or amplify a physiological immune response against senescent cells can provide new perspectives to extend active longevity. In this mini-review, the authors summarize the current understanding of the role of T-cell immunity in the fight against senescent cells and discuss the prospects of stimulating adaptive immunity for combating the accumulation of senescent cells that occurs with age.

Tentaclins-A Novel Family of Phage Receptor-Binding Proteins That Can Be Hypermutated by DGR Systems.

Diversity-generating retroelements (DGRs) are prokaryotic systems providing rapid modification and adaptation of target proteins. In phages, the main targets of DGRs are receptor-binding proteins that are usually parts of tail structures and the variability of such host-recognizing structures enables phage adaptation to changes on the bacterial host surface. Sometimes, more than one target gene containing a hypermutated variable repeat (VR) can be found in phage DGRs. The role of mutagenesis of two functionally different genes is unclear. In this study, several phage genomes that contain DGRs with two target genes were found in the gut virome of healthy volunteers. Bioinformatics analysis of these genes indicated that they encode proteins with different topology; however, both proteins contain the C-type lectin (C-lec) domain with a hypermutated beta-hairpin on its surface. One of the target proteins belongs to a new family of proteins with a specific topology: N-terminal C-lec domain followed by one or more immunoglobulin domains. Proteins from the new family were named tentaclins after TENTACLe + proteIN. The genes encoding such proteins were found in the genomes of prophages and phages from the gut metagenomes. We hypothesized that tentaclins are involved in binding either to bacterial receptors or intestinal/immune cells.

Alu Methylation Patterns in Type 1 Diabetes: A Case-Control Study.

Evidence suggests that genome-wide hypomethylation may promote genomic instability and cellular senescence, leading to chronic complications in people with diabetes mellitus. Limited data are however available on the Alu methylation status in patients with type 1 diabetes (T1D). Methods: We investigated DNA methylation levels and patterns of Alu methylation in the peripheral blood of 36 patients with T1D and 29 healthy controls, matched for age and sex, by using the COmbined Bisulfite Restriction Analysis method (COBRA). Results: Total Alu methylation rate (mC) was similar between patients with T1D and controls (67.3% (64.4-70.9%) vs. 68.0% (62.0-71.1%), p = 0.874). However, patients with T1D had significantly higher levels of the partial Alu methylation pattern (mCuC + uCmC) (41.9% (35.8-45.8%) vs. 36.0% (31.7-40.55%), p = 0.004) compared to healthy controls. In addition, a positive correlation between levels of glycated hemoglobin (HbA1c) and the partially methylated loci (mCuC + uCmC) was observed (Spearman's rho = 0.293, p = 0.018). Furthermore, significant differences were observed between patients with T1D diagnosed before and after the age of 15 years regarding the total methylation mC, the methylated pattern mCmC and the unmethylated pattern uCuC (p = 0.040, p = 0.044 and p = 0.040, respectively). Conclusions: In conclusion, total Alu methylation rates were similar, but the partial Alu methylation pattern (mCuC + uCmC) was significantly higher in patients with T1D compared to healthy controls. Furthermore, this pattern was associated positively with the levels of HbA1c and negatively with the age at diagnosis.

Diversity at single nucleotide to pangenome scales among sulfur cycling bacteria in salt marshes.

IMPORTANCE: Salt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems.

Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia.

Certain classes of genetic variation still escape detection in clinical sequencing analysis. One such class is retroelement insertion, which has been reported as a cause of Mendelian diseases and may offer unique therapeutic implications. Here, we conducted retroelement profiling on whole-genome sequencing data from a cohort of 237 individuals with ataxia telangiectasia (A-T). We found 15 individuals carrying retroelement insertions in ATM, all but one of which integrated in noncoding regions. Systematic functional characterization via RNA sequencing, RT-PCR, and/or minigene splicing assays showed that 12 out of 14 intronic insertions led or contributed to ATM loss of function by exon skipping or activating cryptic splice sites. We also present proof-of-concept antisense oligonucleotides that suppress cryptic exonization caused by a deep intronic retroelement insertion. These results provide an initial systematic estimate of the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as ∼2.1%-5.5%. Our study highlights the importance of retroelement insertions as causal variants and therapeutic targets in genetic diseases.

Lyme Disease Agent Reservoirs Peromyscus leucopus and P. maniculatus Have Natively Inactivated Genes for the High-Affinity Immunoglobulin Gamma Fc Receptor I (CD64).

The abundant and widely distributed deermice Peromyscus leucopus and P. maniculatus are important reservoirs for several different zoonotic agents in North America. For the pathogens they persistently harbor, these species are also examples of the phenomenon of infection tolerance. In the present study a prior observation of absent expression of the high-affinity Fc immunoglobulin gamma receptor I (FcγRI), or CD64, in P. leucopus was confirmed in an experimental infection with Borreliella burgdorferi, a Lyme disease agent. We demonstrate that the null phenotype is attributable to a long-standing inactivation of the Fcgr1 gene in both species by a deletion of the promoter and coding sequence for the signal peptide for FcγRI. The Fcgr1 pseudogene was also documented in the related species P. polionotus. Six other Peromyscus species, including P. californicus, have coding sequences for a full-length FcγRI, including a consensus signal peptide. An inference from reported phenotypes for null Fcgr1 mutations engineered in Mus musculus is that one consequence of pseudogenization of Fcgr1 is comparatively less inflammation during infection than in animals, including humans, with undisrupted, fully active genes.

Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon.

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

Regulatory Role of Insulin on Endogenous L1 ORF1 and NEFM Gene Expression through PI3K Signaling Pathway Specifically in Neuroblastoma Cell Line.

Background: One of the most important endogenous factors causing genomic instability in human cells is L1s retrotransposons. In this study, we assume that increased activity of L1 retrotransposons (specifically L1 expression) might be induced by hyperglycemia and hyperinsulinemia in neuroblastoma cell line. Methods: Two different cell lines (BE (2)-M17 and HEK293) were treated with insulin and its PI3K signaling pathway inhibitor under three conditional media including hyperglycemic and retinoic acid treatment in the department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran in 2018. The expression of L1 ORF1, as well as genes involved in insulin signaling pathway and neuronal stress and structure were measured at RNA level. Results: Insulin could significantly down regulate the expression of L1 ORF1 and NEFM genes. Hyperglycemia result in severe decrease in expression of all candidate genes in control neuroblastoma but not HEK293 cells. Retinoic acid as the concentration used in this study cause increase stemness in neuroblastoma but not HEK293 cells. We could not find significant correlation between expression pattern of other genes tested in our study and L1 ORF1 expression. Conclusion: Total regulatory effect of insulin on L1 ORF1 RNA expression as well as NEFM markedly in BE (2)-M17 cell line. Although these results could not be interpreted as L1 retrotransposition, expression of L1 RNA during stress conditions might be considered following inhibition of the insulin pathway. The result of this study also confirms the impotence of insulin on human evolution.

Conserved microRNAs and Flipons Shape Gene Expression during Development by Altering Promoter Conformations.

The classical view of gene regulation draws from prokaryotic models, where responses to environmental changes involve operons regulated by sequence-specific protein interactions with DNA, although it is now known that operons are also modulated by small RNAs. In eukaryotes, pathways based on microRNAs (miR) regulate the readout of genomic information from transcripts, while alternative nucleic acid structures encoded by flipons influence the readout of genetic programs from DNA. Here, we provide evidence that miR- and flipon-based mechanisms are deeply connected. We analyze the connection between flipon conformation and the 211 highly conserved human miR that are shared with other placental and other bilateral species. The direct interaction between conserved miR (c-miR) and flipons is supported by sequence alignments and the engagement of argonaute proteins by experimentally validated flipons as well as their enrichment in promoters of coding transcripts important in multicellular development, cell surface glycosylation and glutamatergic synapse specification with significant enrichments at false discovery rates as low as 10-116. We also identify a second subset of c-miR that targets flipons essential for retrotransposon replication, exploiting that vulnerability to limit their spread. We propose that miR can act in a combinatorial manner to regulate the readout of genetic information by specifying when and where flipons form non-B DNA (NoB) conformations, providing the interactions of the conserved hsa-miR-324-3p with RELA and the conserved hsa-miR-744 with ARHGAP5 genes as examples.

The Immunological Conundrum of Endogenous Retroelements.

Our defenses against infection rely on the ability of the immune system to distinguish invading pathogens from self. This task is exceptionally challenging, if not seemingly impossible, in the case of retroviruses that have integrated almost seamlessly into the host. This review examines the limits of innate and adaptive immune responses elicited by endogenous retroviruses and other retroelements, the targets of immune recognition, and the consequences for host health and disease. Contrary to theoretical expectation, endogenous retroelements retain substantial immunogenicity, which manifests most profoundly when their epigenetic repression is compromised, contributing to autoinflammatory and autoimmune disease and age-related inflammation. Nevertheless, recent evidence suggests that regulated immune reactivity to endogenous retroelements is integral to immune system development and function, underpinning cancer immunosurveillance, resistance to infection, and responses to the microbiota. Elucidation of the interaction points with endogenous retroelements will therefore deepen our understanding of immune system function and contribution to disease.

Z-RNA biology: a central role in the innate immune response?

Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the immune response. Here, we review how Z-RNA features present in cellular RNAs-especially containing retroelements-could be recognized by a family of winged helix proteins, with an impact on host defense. We also discuss how mutations to specific Z-contacting amino acids disrupt their ability to stabilize Z-RNA, resulting in functional losses. We end by highlighting knowledge gaps in the field, which, if addressed, would significantly advance this active area of research.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution.

BACKGROUND: Endogenous expression of L1 mRNA is the first step in an L1-initiated mutagenesis event. However, the contribution of individual cell types to patterns of organ-specific L1 mRNA expression remains poorly understood, especially at single-locus resolution. We introduce a method to quantify expression of mobile elements at the single-locus resolution in scRNA-Seq datasets called Single Cell Implementation to Find Expressed Retrotransposons (SCIFER). SCIFER aligns scRNA-Seq reads uniquely to the genome and extracts alignments from single cells by cell-specific barcodes. In contrast to the alignment performed using default parameters, this alignment strategy increases accuracy of L1 locus identification by retaining only reads that are uniquely mapped to individual L1 loci. L1 loci expressed in single cells are unambiguously identified using a list of L1 loci manually validated to be expressed in bulk RNA-Seq datasets generated from the same cell line or organ. RESULTS: Validation of SCIFER using MCF7 cells determined technical parameters needed for optimal detection of L1 expression in single cells. We show that unsupervised analysis of L1 expression in single cells exponentially inflates both the levels of L1 expression and the number of expressed L1 loci. Application of SCIFER to analysis of scRNA-Seq datasets generated from mouse and human testes identified that mouse Round Spermatids and human Spermatogonia, Spermatocytes, and Round Spermatids express the highest levels of L1 mRNA. Our analysis also determined that similar to mice, human testes from unrelated individuals share as much as 80% of expressed L1 loci. Additionally, SCIFER determined that individual mouse cells co-express different L1 sub-families and different families of transposable elements, experimentally validating their co-existence in the same cell. CONCLUSIONS: SCIFER detects mRNA expression of individual L1 loci in single cells. It is compatible with scRNA-Seq datasets prepared using traditional sequencing methods. Validated using a human cancer cell line, SCIFER analysis of mouse and human testes identified key cell types supporting L1 expression in these species. This will further our understanding of differences and similarities in endogenous L1 mRNA expression patterns in mice and humans.

Evolution and dosage compensation of nucleolar organizing regions (NORs) mediated by mobile elements in turtles with female (ZZ/ZW) but not with male (XX/XY) heterogamety.

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.

A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus.

Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly silenced by the host genome, they have been found to be activated in multiple disease states, such as auto-inflammatory disorders and neurological diseases. However, the numerous copies in mammalian genomes and the lack of tools to study them make defining their role in health and diseases challenging. In this study, we identified eight copies of the zebrafish endogenous retrovirus zferv. We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single-cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases.

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.

Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3' overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.

Gene-tree misrooting drives conflicts in phylogenomic coalescent analyses of palaeognath birds.

Phylogenomic analyses of ancient rapid radiations can produce conflicting results that are driven by differential sampling of taxa and characters as well as the limitations of alternative analytical methods. We re-examine basal relationships of palaeognath birds (ratites and tinamous) using recently published datasets of nucleotide characters from 20,850 loci as well as 4301 retroelement insertions. The original studies attributed conflicting resolutions of rheas in their inferred coalescent and concatenation trees to concatenation failing in the anomaly zone. By contrast, we find that the coalescent-based resolution of rheas is premised upon extensive gene-tree estimation errors. Furthermore, retroelement insertions contain much more conflict than originally reported and multiple insertion loci support the basal position of rheas found in concatenation trees, while none were reported in the original publication. We demonstrate how even remarkable congruence in phylogenomic studies may be driven by long-branch misplacement of a divergent outgroup, highly incongruent gene trees, differential taxon sampling that can result in gene-tree misrooting errors that bias species-tree inference, and gross homology errors. What was previously interpreted as broad, robustly supported corroboration for a single resolution in coalescent analyses may instead indicate a common bias that taints phylogenomic results across multiple genome-scale datasets. The updated retroelement dataset now supports a species tree with branch lengths that suggest an ancient anomaly zone, and both concatenation and coalescent analyses of the huge nucleotide datasets fail to yield coherent, reliable results in this challenging phylogenetic context.

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase.

Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3' ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.