Small RNA Profiling Research Articles

Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis.

Genome-wide analysis of small RNAs identifies somatic embryogenesis- specific miRNAs and their targets and provides novel insights into the mechanisms governing somatic embryogenesis in coconut, a highly in vitro recalcitrant species. Coconut, a major plantation crop of the tropics is recalcitrant to in vitro culture with a very low rate of somatic embryo turnover. Clonal propagation to enhance the production of high yielding, disease-free planting material in coconut has remained a distant reality. To better understand the molecular basis of this recalcitrance and to throw light on the complex regulatory network involved in the transition of coconut somatic cells to embryogenic calli, genome-wide profiling of small RNAs from embryogenic (EC) and non-embryogenic calli (NEC) was undertaken using Illumina Hiseq 2000 platform. We have identified a total of 110 conserved miRNAs (representing 46 known miRNA families) in both types of calli. In addition, 97 novel miRNAs (48 specific to EC, 21 specific to NEC and 28 common to both the libraries) were also identified. Among the conserved miRNAs, 10 were found to be differentially expressed between NEC and EC libraries with a log2 fold change > 2 following RPM-based normalization. miR156f, miR167c, miR169a, miR319a, miR535a, and miR5179 are upregulated and miR160a, miR166a, miR171a, and miR319b are down-regulated in NEC. To confirm the differential expression pattern and their regulatory role in SE, the expression patterns of miRNAs and their putative targets were analyzed using qRT- PCR and most of the analyzed miRNA-target pairs showed inverse correlation during somatic embryogenesis. Selected targets were further validated by RNA ligase mediated rapid amplification of 5' cDNA ends (5'RLM-RACE). Our data suggest that a few conserved miRNAs and species-specific miRNAs act in concert to regulate the process of somatic embryogenesis in coconut. The results of this study provide the first overview into the regulatory landscape of somatic embryogenesis in coconut and possible strategies for fine-tuning or reprogramming to enhance somatic embryo turn over in coconut.

The Role of microRNAs, Long Non-coding RNAs, and Circular RNAs in Cervical Cancer.

Prolonged infection of uterine cervix epithelium with human papillomavirus (HPV) and constitutive expression of viral oncogenes have been recognized as the main cause of the complex molecular changes leading to transformation of cervical epithelial cells. Deregulated expression of microRNAs (miRNA), long non-coding RNAs (lncRNA), and circular RNAs (circRNA) is involved in the initiation and promotion processes of cervical cancer development. Expression profiling of small RNAs in cervical neoplasia revealed up-regulated “oncogenic” miRNAs, such as miR-10a, miR-21, miR-19, and miR-146a, and down regulated “tumor suppressive” miRNAs, including miR-29a, miR-372, miR-214, and miR-218, associated with cell growth, malignant transformation, cell migration, and invasion. Also several lncRNAs, comprising among others HOTAIR, MALAT1, GAS5, and MEG3, have shown to be associated with various pathogenic processes such as tumor progression, invasion as well as therapeutic resistance and emerged as new diagnostic and prognostic biomarkers in cervical cancer. Moreover, human genes encoded circular RNAs, such as has_circ-0018289, have shown to sponge specific miRNAs and to concur to the deregulation of target genes. Viral encoded circE7 has also demonstrated to overexpress E7 oncoprotein thus contributing to cell transformation. In this review, we summarize current literature on the complex interplay between miRNAs, lncRNAs, and circRNAs and their role in cervical neoplasia.

Human sperm RNA code senses dietary sugar.

A new study reveals that a high-sugar diet acutely alters human sperm small RNA profiles after 1 week and that these changes are associated with changes in sperm motility. This rapid response by sperm to nutritional fluctuation raises intriguing questions regarding the underlying mechanisms and the potential effects on offspring metabolic health.

Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods

ABSTRACTEmbryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media . We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously.Abbreviations: PGT-A: Preimplantation genetic testing for aneuploidies; ECCM: Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles; PCA: Principal component analysis.

Correction for Tarallo et al., “Altered Fecal Small RNA Profiles in Colorectal Cancer Reflect Gut Microbiome Composition in Stool Samples”

Volume 4, no. 5, e00289-19, 2019, [https://doi.org/10.1128/mSystems.00289-19][1]. The following text corrections are necessary. Page 2, third line from the bottom, the paragraph should read: “At the phylum level, the abundances of Proteobacteria and Verrucomicrobia in adenoma patients were

Genome‐wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea

SummaryHybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24‐nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid‐MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down‐regulated hybrid‐MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Tudor-SN Promotes Early Replication of Dengue Virus in the Aedes aegypti Midgut.

SummaryDiseases caused by mosquito-borne viruses have been on the rise for the last decades, and novel methods aiming to use laboratory-engineered mosquitoes that are incapable of carrying viruses have been developed to reduce pathogen transmission. This has stimulated efforts to identify optimal target genes that are naturally involved in mosquito antiviral defenses or required for viral replication. Here, we investigated the role of a member of the Tudor protein family, Tudor-SN, upon dengue virus infection in the mosquito Aedes aegypti. Tudor-SN knockdown reduced dengue virus replication in the midgut of Ae. aegypti females. In immunofluorescence assays, Tudor-SN localized to the nucleolus in both Ae. aegypti and Aedes albopictus cells. A reporter assay and small RNA profiling demonstrated that Tudor-SN was not required for RNA interference function in vivo. Collectively, these results defined a novel proviral role for Tudor-SN upon early dengue virus infection of the Ae. aegypti midgut.

SMETASeq: Combined Profiling of Microbiota and Host Small RNAs

Understanding microbial communities’ roles in human health and disease requires methods that accurately characterize the microbial composition and their activity and effects within human biological samples. We present sMETASeq (small RNA Metagenomics by Sequencing), a novel method that uses sequencing of small RNAs to jointly measure host small RNA expression and create metagenomic profiles. We evaluated the performance of sMETASeq on a mock bacterial community and demonstrated its use on different human samples, including colon cancer, oral leukoplakia, cervix cancer and a panel of human biofluids. In all datasets, the detected microbes reflected the biology of the different sample types.

Small RNA Profiling of piRNAs in Colorectal Cancer Identifies Consistent Overexpression of piR-24000 That Correlates Clinically with an Aggressive Disease Phenotype.

Piwi-interacting RNAs (piRNAs) represent a novel class of small non-coding RNAs (ncRNAs) that have been shown to have a deregulated expression in several cancers, although their clinical significance in colorectal cancer (CRC) remains unclear. With an aim of delineating the piRNA distribution in CRC, we conducted a systematic discovery and validation of piRNAs within two clinical cohorts. In the discovery phase, we profiled tumor and adjacent normal tissues from 18 CRC patients by deep sequencing and identified a global piRNA downregulation in CRC. Moreover, we identified piR-24000 as an unexplored piRNA that was significantly overexpressed in CRC. Using qPCR, we validated the overexpression of piR-24000 in 87 CRC patients. Additionally, we identified a significant association between a high expression of piR-24000 and an aggressive CRC phenotype including poor differentiation, presence of distant metastases, and a higher stage. Lastly, ROC analysis demonstrated a strong diagnostic power of piR-24000 in discriminating CRC patients from normal subjects. Taken together, this study provides one of the earliest large-scale reports of the global distribution of piRNAs in CRC. In addition, piR-24000 was identified as a likely oncogene in CRC that can serve as a biomarker or a therapeutic target.

Analysis of miRNAs in Two Wheat Cultivars Infected With Puccinia striiformis f. sp. tritici.

MicroRNAs are small RNAs that regulate gene expression in eukaryotes. In this study, we analyzed the small RNA profiles of two cultivars that exhibit different reactions to stripe rust infection: one susceptible, the other partially resistant. Using small RNA libraries prepared from the two wheat cultivars infected with stripe rust fungus (Puccinia striiformis f. sp. tritici), we identified 182 previously known miRNAs, 91 variants of known miRNAs, and 163 candidate novel wheat miRNAs. Known miRNA loci were usually copied in all three wheat sub-genomes, whereas novel miRNA loci were often specific to a single sub-genome. DESeq2 analysis of differentially expressed microRNAs revealed 23 miRNAs that exhibit cultivar-specific differences. TA078/miR399b showed cultivar-specific differential regulation in response to infection. Using different target prediction algorithms, 145 miRNAs were predicted to target wheat genes, while 69 miRNAs were predicted to target fungal genes. We also confirmed reciprocal expression of TA078/miR399b and tae-miR9664 and their target genes in different treatments, providing evidence for miRNA-mediated regulation during infection. Both known and novel miRNAs were predicted to target fungal genes, suggesting trans-kingdom regulation of gene expression. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the yet-uncharacterized roles for miRNAs in the wheat-stripe rust pathosystem.

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

ABSTRACT Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Osteoclast-derived apoptotic bodies show extended biological effects of parental cell in promoting bone defect healing.

Apoptotic bodies (ABs) traditionally considered as garbage bags that enclose residual components of dead cells are gaining increasing attentions due to their potential roles in intercellular communications. In bone turn over, at the end of bone resorption phase, most osteoclasts undergo apoptosis, generating large amounts of ABs. However, it remains unclear of the role of osteoclast-derived ABs in bone remodeling.Methods: Staurosporine (STS) was used to apoptotic induction and differential centrifugation was used to isolate ABs. Western blotting, flowcytometry and Transmission electron microscopy (TEM) were performed for ABs identification, while whole transcriptome of ABs from osteoclasts at different stages was detected by RNA-seq. VENN analysis and gene set enrichment analysis (GSEA) were performed to compare the profile similarities between ABs and parental cells. In vitro efficacy of ABs on angiogenesis and osteogenesis were evaluated by tube formation assay and ALP staining. In vivo, calvarial defect mice model was used to assess the effects of ABs-modified decalcified bone matrix (DBM) scaffolds on angiogenesis and osteogenesis.Results: Here we mapped the whole transcriptome paralleled with small RNA profiling of osteoclast derived ABs at distinct differentiation stages. Whole transcriptome analysis revealed significant differences in RNA signatures among the ABs generated from osteoclasts at different stages. By comparing with parental osteoclast RNA profiles, we found that the transcriptome of ABs exhibited high similarities with the corresponding parental cells. Functionally, in vitro and in vivo studies showed that similar with the parental cells, pOC-ABs potentiated endothelial progenitor cell proliferation and differentiation, whereas mOC-ABs promoted osteogenic differentiation. The inherited biological effects of ABs were shown mediated by several enriched lncRNAs of which the interference abolished AB functions.Conclusions: Our study revealed the total RNA profiles of osteoclast derived ABs and demonstrated their biological functions. Both gene set and functional analysis indicated that osteoclast derived ABs are biologically similar with the parental cells suggesting their bridging role in osteoclast-osteoblast coupling in bone remodeling.

Small noncoding RNA discovery and profiling with sRNAtools based on high-throughput sequencing.

Small noncoding RNAs (sRNA/sncRNAs) are generated from different genomic loci and play important roles in biological processes, such as cell proliferation and the regulation of gene expression. Next-generation sequencing (NGS) has provided an unprecedented opportunity to discover and quantify diverse kinds of sncRNA, such as tRFs (tRNA-derived small RNA fragments), phasiRNAs (phased, secondary, small-interfering RNAs), Piwi-interacting RNA (piRNAs) and plant-specific 24-nt short interfering RNAs (siRNAs). However, currently available web-based tools do not provide approaches to comprehensively analyze all of these diverse sncRNAs. This study presents a novel integrated platform, sRNAtools (https://bioinformatics.caf.ac.cn/sRNAtools), that can be used in conjunction with high-throughput sequencing to identify and functionally annotate sncRNAs, including profiling microRNAss, piRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and rRNAs and discovering isomiRs, tRFs, phasiRNAs and plant-specific 24-nt siRNAs for up to 21 model organisms. Different modules, including single case, batch case, group case and target case, are developed to provide users with flexible ways of studying sncRNA. In addition, sRNAtools supports different ways of uploading small RNA sequencing data in a very interactive queue system, while local versions based on the program package/Docker/virtureBox are also available. We believe that sRNAtools will greatly benefit the scientific community as an integrated tool for studying sncRNAs.

IMIRAGE: an R package to impute microRNA expression using protein-coding genes.

MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Due to challenges in accurate profiling of small RNAs, a vast majority of public transcriptome datasets lack reliable miRNA profiles. However, the biological consequence of miRNA activity in the form of altered protein-coding gene (PCG) expression can be captured using machine-learning algorithms. Here, we present iMIRAGE (imputed miRNA activity from gene expression), a convenient tool to predict miRNA expression using PCG expression of the test datasets. The iMIRAGE package provides an integrated workflow for normalization and transformation of miRNA and PCG expression data, along with the option to utilize predicted miRNA targets to impute miRNA activity from independent test PCG datasets. The iMIRAGE package for R, along with package documentation and vignette, is available at https://aritronath.github.io/iMIRAGE/index.html. Supplementary data are available at Bioinformatics online.

Identification of miRNA Targets by AtFT Overexpression in Tobacco

The onset of flowering is regulated by complex gene networks that integrate multiple genetic cues to floral transition in plants. The highly conserved florigen FLOWERING LOCUS T (FT) functions at the core of this process. Here, we demonstrate that overexpression of Arabidopsis FT in tobacco (oxAtFT) leads to precocious flowering in a photoperiod-independent manner. Strikingly altered phenotypes such as stunted growth, increased number of axillary branches, change in leaf morphology, and elongated style were observed in oxAtFT transgenic tobacco plants. In addition, the generation time of oxAtFT tobacco was drastically reduced by 8–10 weeks. We observed defects in lateral root formation in both oxAtFT tobacco and oxFT Arabidopsis lines. Interestingly, oxAtFT tobacco displayed significant heat and drought tolerance as compared to the wild type. Through small RNA profiling, we observed the upregulation of a conserved microRNA (nta-miR393a-5p) of miR393 family that targets the TRANSPORT INHIBITOR RESPONSE 1-like (TIR1) gene. Overall, the heterologous expression of AtFT in tobacco may have potential in imparting abiotic stress tolerance in other plant species through plant biotechnology–based approaches.

Small RNA profiling from meiotic and post-meiotic anthers reveals prospective miRNA-target modules for engineering male fertility in sorghum

Understanding male gametophyte development is essential to augment hybrid production in sorghum. Although small RNAs are known to critically influence anther/pollen development, their roles in sorghum reproduction have not been deciphered yet. Here, we report small RNA profiling and high-confidence annotation of microRNAs (miRNAs) from meiotic and post-meiotic anthers in sorghum. We identified 262 miRNAs (82 known and 180 novel), out of which 58 (35 known and 23 novel) exhibited differential expression between two stages. Out of 35 differentially expressed known miRNAs, 13 are known to regulate anther/pollen development in other plant species. We also demonstrated conserved spatiotemporal patterns of 21- and 24-nt phasiRNAs and their respective triggers, miR2118 and miR2275, in sorghum anthers as evidenced in other monocots. miRNA target identification yielded 5622 modules, of which 46 modules comprising 16 known and 8 novel miRNA families with 38 target genes are prospective candidates for engineering male fertility in grasses.

Rice Dwarf Virus Small RNA Profiles in Rice and Leafhopper Reveal Distinct Patterns in Cross-Kingdom Hosts.

RNA silencing has evolved as a widespread antiviral strategy in many eukaryotic organisms. Antiviral RNA silencing is mediated by virus-derived small RNAs (vsiRNAs), created by the cleavage of double-stranded viral RNA substrates by Dicer (Dcr) in animals or Dicer-like (DCL) proteins in plants. However, little is known about how the RNA silencing mechanisms of different hosts respond to the same virus infection. We performed high-throughput small RNA sequencing in Nephotettix cincticeps and Oryza sativa infected with Rice dwarf phytoreovirus and analyzed the distinct accumulation of vsiRNAs in these two hosts. The results suggested a potential branch in the evolution of antiviral RNA silencing of insect and plant hosts. The rice vsiRNAs were predominantly 21 and 22 nucleotides (nt) long, suggesting that OsDCL4 and OsDCL2 are involved in their production, whereas 21-nt vsiRNAs dominated in leafhopper, suggesting the involvement of a Dcr-2 homolog. Furthermore, we identified ~50-fold more vsiRNAs in rice than in leafhoppers, which might be partially attributable to the activity of RNA-dependent RNA polymerase 6 (RDR6) in rice and the lack of RDR genes in leafhoppers. Our data established a basis for further comparative studies on the evolution of RNA silencing-based interactions between a virus and its hosts, across kingdoms.

3D cell culture stimulates the secretion of in vivo like extracellular vesicles

For studying cellular communications ex-vivo, a two-dimensional (2D) cell culture model is currently used as the “gold standard”. 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to represent in vivo extracellular matrix (ECM) microenvironment. Our study observed that 2D- culture derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D- culture derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D- culture derived EV small RNA profile exhibited a much higher similarity (~96%) to in vivo circulating EVs derived from cervical cancer patient plasma. However, 2D- culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimicking in vivo tissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.

Hybrid Decay: A Transgenerational Epigenetic Decline in Vigor and Viability Triggered in Backcross Populations of Teosinte with Maize.

In the course of generating populations of maize with teosinte chromosomal introgressions, an unusual sickly plant phenotype was noted in individuals from crosses with two teosinte accessions collected near Valle de Bravo, Mexico. The plants of these Bravo teosinte accessions appear phenotypically normal themselves and the F1 plants appear similar to typical maize × teosinte F1s. However, upon backcrossing to maize, the BC1 and subsequent generations display a number of detrimental characteristics including shorter stature, reduced seed set, and abnormal floral structures. This phenomenon is observed in all BC individuals and there is no chromosomal segment linked to the sickly plant phenotype in advanced backcross generations. Once the sickly phenotype appears in a lineage, normal plants are never again recovered by continued backcrossing to the normal maize parent. Whole-genome shotgun sequencing reveals a small number of genomic sequences, some with homology to transposable elements, that have increased in copy number in the backcross populations. Transcriptome analysis of seedlings, which do not have striking phenotypic abnormalities, identified segments of 18 maize genes that exhibit increased expression in sickly plants. A de novo assembly of transcripts present in plants exhibiting the sickly phenotype identified a set of 59 upregulated novel transcripts. These transcripts include some examples with sequence similarity to transposable elements and other sequences present in the recurrent maize parent (W22) genome as well as novel sequences not present in the W22 genome. Genome-wide profiles of gene expression, DNA methylation, and small RNAs are similar between sickly plants and normal controls, although a few upregulated transcripts and transposable elements are associated with altered small RNA or methylation profiles. This study documents hybrid incompatibility and genome instability triggered by the backcrossing of Bravo teosinte with maize. We name this phenomenon "hybrid decay" and present ideas on the mechanism that may underlie it.

Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3' trailer-derived tRNA fragments.

tRNA-derived RNA fragments (tRFs) have emerged as a new class of functional RNAs implicated in cancer, metabolic and neurological disorders, and viral infection. Yet our understanding of their biogenesis and functions remains limited. In the present study, through analysis of small RNA profile we have identified a distinct set of tRFs derived from pre-tRNA 3′ trailers in the hepatocellular carcinoma cell line Huh7. Among those tRFs, tRF_U3_1, which is a 19-nucleotide-long chr10.tRNA2-Ser(TGA)-derived trailer, was expressed most abundantly in both Huh7 and cancerous liver tissues, being present primarily in the cytoplasm. We show that genetic loss of tRF_U3_1 does not affect cell growth and it is not involved in Ago2-mediated gene silencing. Using La/SSB knockout Huh7 cell lines, we demonstrate that this nuclear-cytoplasmic shuttling protein directly binds to the 3′ U-tail of tRF_U3_1 and other abundantly expressed trailers and plays a critical role in their stable cytoplasmic accumulation. The pre-tRNA trailer-derived tRFs capable of sequestering the limiting amounts of La/SSB in the cytoplasm rendered cells resistant to various RNA viruses, which usurp La/SSB with RNA chaperone activity for their gene expression. Collectively, our results establish the trailer-derived tRF-La/SSB interface, regulating viral gene expression.