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At least 19 recordsLinked to original sources
PubMed2026

A modular class-aware workflow for small RNA sequencing analysis using mouse sperm as a case study.

BACKGROUND: Small RNA sequencing analysis is challenging because RNA classes differ in biogenesis, sequence redundancy, genomic organization, and annotation reliability. Integrated workflows accommodating these constraints remain limited, particularly for fragment-level and cluster-level analysis. METHODS: We present a reproducible, containerized, class-aware workflow for small RNA sequencing analysis, using mouse sperm as a case study. The workflow combines standardized preprocessing with complementary annotation and quantification strategies for microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), ribosomal RNA-derived small RNAs (rsRNAs), and PIWI-interacting RNA (piRNA)-enriched genomic clusters. Using sperm small RNA data from offspring of lipopolysaccharide (LPS)-exposed male mice, we compared integrated-reference mapping, multi-class annotation, fragment-level tsRNA profiling, and genome-based piRNA cluster analysis, with custom modules for locus-aware harmonization and condition-specific cluster analysis. RESULTS: Integrated-reference mapping aligned 88.17% of reads and retained 690 features after filtering. It identified 11 differentially expressed miRNAs between LPS and controls, while other classes showed limited signal. Fragment-level profiling improved tsRNA resolution. piRNA cluster analysis identified 958 control and 940 LPS clusters, with 18 control-specific and no LPS-specific clusters. CONCLUSION: This workflow supports transparent, reproducible, class-aware interpretation of small RNA sequencing data while emphasizing cautious interpretation of piRNA-enriched signals from total small RNA sequencing.

Small non-coding RNA analysis↗
PubMed2025

A hypoxia-responsive tRNA-derived small RNA confers renal protection through RNA autophagy.

Transfer RNA-derived small RNAs (tsRNAs or tDRs) perform a range of cellular functions. Here, we showed that tRNA-Asp-GTC-3'tDR, a hypoxia-induced tDR derived from the 3' end of tRNA-Asp-GTC, activated autophagic flux in kidney cells and its silencing blocked autophagic flux. Functional gain-/loss-of-function studies in murine kidney disease models demonstrated a substantial renoprotective function of tRNA-Asp-GTC-3'tDR. Mechanistically, tRNA-Asp-GTC-3'tDR assembled stable G-quadruplex structures and sequestered pseudouridine synthase 7 (PUS7), preventing catalytic pseudouridylation of histone mRNAs. The resulting pseudouridylation deficiency directed histone mRNAs to the autophagosome-lysosome pathway, triggering RNA autophagy. This tDR-induced RNA autophagy pathway was activated during murine and human kidney diseases, suggesting clinical relevance. Thus, tRNA-Asp-GTC-3'tDR plays a role in regulating RNA autophagy, which helps to maintain homeostasis in kidney cells and protects against kidney injury.

Animals↗
PubMed1979

Synthesis of a small RNA in cells coinfected by standard and defective interfering particles of vesicular stomatitis virus.

A small RNA, containing approximately 50 nucleotides, is synthesized by cells coinfected with standard vesicular stomatitis virus and its defective interfering (DI) particles. Infection of cells by standard virus or DI particles alone does not lead to synthesis of significant amounts of small RNA. The RNA is initiated at its 5' end with (p)ppXp and is not polyadenylylated at the 3' end despite a content of 51% adenosine. It has sequences complementary to the genome of a DI particle. The synthesis of the small RNA correlates with the replication of the genome of DI particles with molar ratio small RNA/genome RNA of DI particles greater than 50. When replication of DI genomes is prevented by the addition of cycloheximide or prior UV irradiation of DI particles, small RNA is not synthesized in coinfected cells. These results indicate that the small RNA is not the result of transcriptional initiation and that it may relate to interference mediated by DI particles.

Animals↗
PubMed2026

Invertebrate miRNA pva-small RNA-11881/pva-miR-11881 as a potential RNA-based therapeutic against white spot syndrome virus in infected shrimp.

Small RNAs and microRNAs (miRNAs) play diverse roles in host virus interactions and hold promise for therapeutic applications. An uncharacterized shrimp miRNA with potent activity against white spot syndrome virus (WSSV), a major double-stranded DNA pathogen in aquaculture, was identified and characterized. Among the 1,239 differentially expressed unannotated small RNAs in Penaeus vannamei hemocytes, one of the most strongly downregulated candidates, termed pva-small RNA-11881 or pva-miR-11881, was predicted to target multiple WSSV genes. A pva-small RNA-11881/pva-miR-11881 isomir that originates from the 5' untranslated region of a host lipase 3-like gene was identified. Its primary transcript contains Drosha and Dicer processing sites, and the precursor exhibits canonical pre-miRNA features. In vivo administration of its primary transcript, pva-pri-miR-11881, significantly reduced WSSV copy number and improved shrimp survival. Mechanistically, pva-miR-11881 directly suppresses crucial WSSV genes WSSV004, WSSV164, and WSSV419 and modulates the host immune response against WSSV infection by enhancing phenoloxidase activity, thereby reducing apoptosis and necrosis, and promoting caspase-1-mediated cell death. These findings reveal that the pva-miR-11881 in P. vannamei holds strong potential as a biotherapeutic agent for managing viral diseases in shrimp.

Animals↗
PubMed2026

Systematic identification of germ granule proteins reveals specialized roles in RNAi and small RNA inheritance.

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. However, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has coevolved with PRG-1 across clade V nematodes, with a conserved structured N terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily coadapted PIWI interactor critical for siRNA biogenesis.

Animals↗
PubMed2026

When light colour matters: Spectral quality is associated with distinct small RNA candidates in Arabidopsis thaliana.

The spectral composition of incident light is perceived by plant photoreceptors and can rapidly reshape downstream gene expression programs; however, its impact on the small-RNA layer beyond annotated miRNAs remains incompletely characterized. The objective of this study was to determine whether 3 h exposure of Arabidopsis thaliana rosettes to blue, green, red, or white light at an equal photosynthetic photon flux density (PPFD) of 400 μmol m-2 s-1 is associated with distinct profiles of candidate small RNAs. A stringent discovery and post-processing workflow was applied to identify high-confidence, treatment-associated small RNA candidates beyond annotated miRNA. The miRkwood-based pipeline, combined with additional filtering and contextual annotation, recovered a compact set of candidates dominated by the blue-light treatment (41 candidates), compared with fewer candidates associated with red (8), green (6), and white light (3). Genomic-context analysis indicated that most candidate sites were intergenic, with candidates detected under green light being entirely intergenic, and overlap with transposon annotations was used to distinguish candidates arising from transposon-rich genomic space. Sequence-feature profiling revealed pronounced treatment-dependent terminal nucleotide biases at both 5' and 3' ends, consistent with spectrum-associated shifts in length-class and terminal-nucleotide signatures that are informative for AGO-loading hypotheses. Target prediction highlighted a subset of genes showing convergent targeting by multiple independent blue-associated candidates, and duplex summaries showed structured, plant-like complementarity patterns (including frequent 10-11 pairing). Together, these results indicate that 3 h exposure to wavelength-defined light at 400 μmol m-2 s-1, particularly blue light, is associated with a distinct profile of detectable candidate small RNAs in Arabidopsis leaves and identify candidate interactions for follow-up validation.

5′ nucleotide bias↗
PubMed2025

A Small RNA Derived From the 5' End of the IS200 tnpA Transcript Regulates Multiple Virulence Regulons in Salmonella typhimurium.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its prevalence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small, highly structured RNA (5'tnpA) that participates in the posttranscriptional control of invF expression, encoding a key transcription factor in this enteropathogen's invasion regulon. To further examine the scope of 5'tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq, revealing the differential expression of over 200 genes in a Salmonella SL1344 5'tnpA disruption strain. This includes the genes for the master regulators of both invasion and flagellar regulons (HilD and FlhDC, respectively), plus genes involved in cysteine biosynthesis and an operon (phsABC) encoding a thiosulfate reductase complex. These expression changes were accompanied by an 80-fold increase in Salmonella invasion of HeLa cells. Follow-up experimentation suggested an additional direct target of 5'tnpA to be the small RNA PinT, which has previously been shown to be a negative regulator of invasion genes through its inhibitory action on key transcription factors governing the Salmonella pathogenicity island 1 regulon. This study provides a powerful new example of bacterial transposon domestication that is based not on the production/use of a regulatory protein or regulatory DNA sequences, but on the function of a transposon-derived small RNA.

Salmonella typhimurium↗
PubMed2026

Phasis: a software tool for register-resolved discovery of plant phased small RNA loci.

Plant PHAS locus discovery remains challenging because phasiRNA-producing loci must be distinguished from other sRNA-producing regions with high abundance or apparent periodicity. This problem is especially acute for reproductive 24-PHAS loci, which occur within genomes that also produce abundant 24-nt siRNAs from nonPHAS regions. We present Phasis, an open-source Python software tool for plant PHAS-locus discovery from small RNA sequencing data. Phasis combines statistical evidence for phased accumulation with locus-level features and a Register-Resolved Locus Interpretation Layer that evaluates whether candidate loci show coherent phased architecture. Across diverse plant datasets, Phasis recovered validated or annotated 21- and 24-PHAS loci with a strong balance between call-level precision and reference-locus recall, and generally outperformed PhaseTank and ShortStack in matched benchmark analyses. The register-resolved interpretation layer reduced unsupported calls by separating coherent phased loci from ambiguous sRNA-producing regions. In maize dcl5 mutant libraries, Phasis showed strong depletion of 24-PHAS recovery, supporting DCL5-dependent recovery of reproductive 24-PHAS signal. Together, these results support Phasis as a biologically interpretable tool for large-scale discovery of plant DCL-dependent phasiRNA loci.

bioinformatics↗
PubMed2026

Identification of Freezing-Responsive microRNAs and Their Targets in Chinese Jujube by Small RNA and Degradome Sequencing.

The jujube tree fruit remains a primary fruit in northern China, yet its geographical distribution and yield are significantly constrained by freezing stress during winter. Numerous studies have highlighted the pivotal regulatory function of microRNAs (miRNAs) in plant responses to low-temperature stress. Nevertheless, the specific miRNAs involved in the response to low temperatures and their associated gene networks in Ziziphus jujuba Mill are not well understood. In this investigation, we utilized high-throughput sequencing to analyze small RNA libraries from branches subjected to temperatures of 4 °C and -30 °C. Our analysis identified a total of 342 miRNAs, comprising 123 known miRNAs and 219 novel miRNAs. The differential expression analysis revealed that under low-temperature conditions, 177 miRNAs underwent significant changes. Among them, specific upregulation of miR319 in the less cold-resistant variety and miR6483 in sensitive variety was observed. By employing degradome sequencing, we identified a total of 1551 target genes corresponding to 3059 unique miRNA target interaction pairs involving 299 miRNAs. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these target genes are primarily associated with transcriptional regulation, metabolic pathways, and genetic information processing. Through a comprehensive analysis, we pinpointed 11 genes corresponding to 9 miRNAs that are implicated in jujube tree cold stress, and 7 target genes of 7 miRNAs were confirmed by 5'-RACE analysis. These miRNAs are likely to exert crucial regulatory functions in the context of jujube tree cold stress. This study is the first to systematically identify miRNAs and their target genes in the response of Ziziphus jujuba Mill to low-temperature stress, which provides important resources for in-depth analysis of the molecular mechanism of jujube tree cold resistance and for cold-resistant breeding.

Ziziphus↗
PubMed2025

The Salmonella pathogenicity island 1-encoded small RNA InvR mediates post-transcriptional feedback control of the activator HilA in Salmonella.

UNLABELLED: Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. Transcription of hilA is controlled by the AraC-like proteins HilD, HilC, and RtsA. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. We used rGRIL-seq (reverse global sRNA target identification by ligation and sequencing) to identify sRNAs that bind to the hilA 5' UTR. The rGRIL-seq data, along with genetic analyses, demonstrate the SPI1-encoded sRNA invasion gene-associated RNA (InvR) base pairs at a site overlapping the hilA ribosome binding site. HilD and HilC activate both invR and hilA. InvR, in turn, negatively regulates the translation of the hilA mRNA. Thus, the SPI1-encoded sRNA InvR acts as a negative feedback regulator of SPI1 expression. Our results suggest that InvR acts to fine-tune SPI1 expression and prevents overactivation of hilA expression, highlighting the complexity of sRNA regulatory inputs controlling SPI1 and Salmonella virulence. IMPORTANCE: Salmonella Typhimurium infections pose a significant public health concern, leading to illnesses that range from mild gastroenteritis to severe systemic infection. Infection requires a complex apparatus that the bacterium uses to invade the intestinal epithelium. Understanding how Salmonella regulates this system is essential for addressing these infections effectively. Here, we show that the small RNA (sRNA) InvR imposes a negative feedback regulation on the expression of the invasion system. This work underscores the role of sRNAs in Salmonella's complex regulatory network, offering new insights into how these molecules contribute to bacterial adaptation and pathogenesis.

Genomic Islands↗
PubMed1976

Serological relations between twelve small RNA viruses of insects.

Serological tests were done to examine the relationships between twelve picorna-like viruses of insects. The results of the tests indicated that the majority of the viruses are unrelated. However, cricket paralysis virus, isolated from Australian wild field crickets, appeared to be identical to Drosophila C virus, independently isolated in France. Cricket paralysis virus was infective for adults of Drosophila melanogaster and its infectivity towards Galleria melonella was neutralised by Drosophila C virus antiserum. It is therefore concluded that cricket paralysis virus and Drosophila C virus are very closely related if not identical.

Animals↗
PubMed1979

Origin of small RNA in von Magnus particles of influenza virus.

A clone of recombinant virus obtained from the cross between WSN and Hong Kong strains of influenza virus gave rise to progeny containing predominantly von Magnus particles. In the electropherogram of virus RNA, the P3 gene was markedly diminished, and a new species of RNA (extra RNA) was present in addition to eight gene segments. The origin of the extra RNA was studied by two-dimensional gel electrophoresis of T1 RNase-generated oligonucleotides. Four out of five large oligonucleotide spots present in the extra RNA matched to those contained by the P3 gene. It was concluded that the extra RNA was derived from the P3 gene probably by deletion. The possible origin of the spot which was present in the extra RNA but not in eight gene segments including P3 was discussed.

Defective Viruses↗
PubMed1979

Physicochemical characterization of a small RNA virus associated with baculovirus infection in Trichoplusia ni.

An RNA virus was isolated from Trichoplusia ni larvae infected with inoculum of the baculovirus of Autographa californica. Physicochemical characterization has revealed a 35-nm virus with a density in CsCl of 1.3 g/cm3 and a sedimentation coefficient of 200S. The virus consisted of 10--15% single-stranded RNA with an apparent molecular weight of 1.9 x 10(6) and coat protein with a subunit size of 67,000. The virus is similar to the Nudaurelia capensis beta virus and also possesses some interesting similarities to the mammalian caliciviruses. The proposed cryptogram of the virus is R/1:1.9/12:S/S:I/O.

Animals↗
PubMed2026

Naturally occurring mutations in replication proteins of a small RNA virus that alter the number, sizes, and relative abundances of subgenomic RNAs.

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in infected cells. sgRNAs are synthesized by virus-encoded replication proteins (RPs), but whether RPs regulate the number and sizes of sgRNAs remains largely unknown. We report multiple naturally occurring mutations within the RPs of turnip crinkle virus (TCV) that alter the number, sizes, and relative abundances of TCV sgRNAs. TCV is a (+) RNA virus that normally produces two sgRNAs: the 1,724-nucleotide (nt) sgRNA1 expressing movement proteins, and the 1,449-nt sgRNA2 expressing capsid protein. A single amino acid change, A113V, within a region shared by TCV RPs p28 and p88, diminished sgRNA1 levels and delayed viral systemic spread. Interestingly, three second-site RP mutations emerged in infected plants that, alone or in combination with A113V, resulted in over-production of sgRNA1 or accumulation of two alternative sgRNAs of 1,876 and 1,601 nt, and rescued A113V defects. The alternative sgRNAs originated from nearly identical recombination events, their size difference reflecting varying 5' extensions. They may have accumulated to high levels through selective stabilization of their (-)-strand intermediates that were in turn derived from transcriptional pausing and recombination. Our findings reveal previously unrecognized constraints on viral RPs that ensure production of sgRNAs with precise sizes and abundances.

Subgenomic RNA↗
PubMed2026

tRNA methylation: functional insights and epitranscriptomic regulation.

tRNAs, one of the most conserved and abundant RNAs, are central components of protein synthesis, transferring genetic information from DNA to proteins through a precise base-pairing mechanism. Post-transcriptional modifications of tRNAs by tRNA modifying enzymes are essential for maintaining their normal physiological functions, including methylation, isomerization and glycosylation. tRNA methylation, particularly 1-methyladenosine (m1A), 5-methylcytidine (m5C), and 7-methylguanosine (m7G), are among the most abundant and diverse types of post-transcriptional modifications of tRNA, which promote the stability of tRNA secondary and tertiary structures and allow for proper translation. In addition, tRNA methylation affects the production and function of tsRNA (tRNA-derived small RNA), small fragments of RNA that further regulate gene expression and protein synthesis. In our review, we discuss the relevant biological functions of tRNA methylation, including tRNA stability, protein translation, and tsRNA biogenesis.

RNA, Transfer↗
PubMed1977

Comparative neutron small-angle scattering study of small spherical RNA viruses.

Small-angle neutron scattering from solutions of small RNA viruses has been used to study protein-nucleic acid organisation. In the five viruses investigated the RNA is confined to a sphere of about 100 A radius, with a central hole (with one possible exception). The interpenetration of RNA and protein varies with viruses and seems to be related to the natrue of the forces stabilising the virus.

Coliphages↗
PubMed1976

Particular small size RNA and RNA fragments from different origins as tumor inducing agents in Datura stramonium.

Particular RNA fragments obtained by action of pancreatic ribonuclease on purified RNAs originating from species totally unrelated to Agrobacterium tumefaciens (Escherichia coli, rabbit, monkey) are capable of inducing the formation of transplantable tumorous tissue when introduced at wounded sites in inverted stems of Datura stramonium maintained under axenic conditions on a medium containing auxin and kinetin. Reovirus RNA and a small size RNA (5-6S) isolated from RNA bound RNA directed DNA polymerase from Escherichia coli also induced the appearance of tumorous tissues which grow on solid synthetic medium in the absence of auxin and kinetin.

Animals↗