poly(UG)-tailed RNAs in genome protection and epigenetic inheritance.

Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.

ZSP-1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans.

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.

Advances and opportunities in RNA structure experimental determination and computational modeling.

Beyond transferring genetic information, RNAs are molecules with diverse functions that include catalyzing biochemical reactions and regulating gene expression. Most of these activities depend on RNAs' specific structures. Therefore, accurately determining RNA structure is integral to advancing our understanding of RNA functions. Here, we summarize the state-of-the-art experimental and computational technologies developed to evaluate RNA secondary and tertiary structures. We also highlight how the rapid increase of experimental data facilitates the integrative modeling approaches for better resolving RNA structures. Finally, we provide our thoughts on the latest advances and challenges in RNA structure determination methods, as well as on future directions for both experimental approaches and artificial intelligence-based computational tools to model RNA structure. Ultimately, we hope the technological advances will deepen our understanding of RNA biology and facilitate RNA structure-based biomedical research such as designing specific RNA structures for therapeutics and deploying RNA-targeting small-molecule drugs.

PhasiHunter: a robust phased siRNA regulatory cascade mining tool based on multiple reference sequences.

SUMMARY: In recent years, phased small interfering RNA has been found to play crucial roles in many biological processes in plants. However, efficiently predicting phasiRNA regulatory cascades with computational methods is still challenging. Here, we introduce PhasiHunter, a phasiRNA regulatory network prediction tool that has several distinctive features compared to existing tools: (i) PhasiHunter employs two major phasiRNA prediction algorithms, namely phase score and hypergeometric distribution-based methods, to ensure the integrity and accuracy of prediction; (ii) PhasiHunter can identify phasiRNAs and their regulatory networks based on multiple reference sequences and the predicted results can be automatically integrated; (iii) PhasiHunter can efficiently identify the phasiRNAs generated through alternative splicing events; and (iv) the excellent data structure and parallel computing architecture allow PhasiHunter to predict phasiRNAs and their regulatory pathways with high efficiency. AVAILABILITY AND IMPLEMENTATION: PhasiHunter is an open-source tool that is available at https://github.com/HuangLab-CBI/PhasiHunter.

RiceNCexp: a rice non-coding RNA co-expression atlas based on massive RNA-seq and small-RNA seq data.

Non-coding RNAs (ncRNAs) play important roles in regulating expression of protein-coding genes. Although gene expression databases have emerged in a timely manner, a comprehensive expression database for ncRNAs is still lacking. Herein, we constructed a rice ncRNA co-expression atlas (RiceNCexp), based on 491 RNA-seq and 274 small RNA (sRNA)-seq datasets. RiceNCexp hosts four types of ncRNAs, namely lncRNAs, PHAS genes, miRNAs, and phasiRNAs. RiceNCexp provides comprehensive expression information for rice ncRNAs in 22 tissues/organs, an efficient tau-based mining tool for tissue-specific ncRNAs, and the robust co-expression analysis among ncRNAs or between ncRNAs and protein-coding genes, based on 116 pairs of RNA-seq and sRNA-seq libraries from the same experiments. In summary, RiceNCexp is a user-friendly and comprehensive rice ncRNA co-expression atlas and can be freely accessed at https://cbi.njau.edu.cn/RiceNCexp/.

PhasiRNAnalyzer: an integrated analyser for plant phased siRNAs.

Phased siRNAs (phasiRNAs) are a class of small interfering RNAs (siRNAs) which play essential roles in plant development and defence. However, only a few phasiRNAs have been extensively studied due to the difficulties in identifying and characterizing plant phasiRNAs by plant biologists. Herein, we describe a comprehensive and multi-functional web server termed PhasiRNAnalyzer, which is able to identify all crucial components in plant phasiRNA's regulatory pathway (phase-initiator→PHAS gene→phasiRNA cluster→target gene). Currently, PhasiRNAnalyzer exhibits the following advantages: I) It is the most comprehensive platform which hosts 170 plant species with 256 genome data, 438 cDNA data and 271 degradome data. II) It can identify all crucial components in phasiRNA's regulatory pathway, and verify the interactions between phasiRNAs and their target genes based on degradome data. III) It can perform differential expression analysis of phasiRNAs on each PHAS gene locus between different samples conveniently. IV) It provides the user-friendly interfaces and introduces several improvements, primarily by making more accurate and efficient analysis when dealing with deep sequencing data. In summary, PhasiRNAnalyzer is a comprehensive and systemic phasiRNA analysis server with high sensitivity and efficiency. It can be freely accessed at https://cbi.njau.edu.cn/PPSA/.

WPMIAS: Whole-degradome-based Plant MicroRNA-Target Interaction Analysis Server.

A critical aspect for exploring the biological function of a microRNA (miRNA) lies on exact detection and validation of its target mRNAs. However, no convenient and efficient web-based server is available for plant biologists to identify the experimentally verified target mRNAs of miRNAs. In this work, we built a comprehensive web-based platform for miRNA-target analysis, named as Whole-degradome-based Plant MiRNA-target Interaction Analysis Server (WPMIAS), for validation of predicted interactions of miRNAs and their target mRNAs (MTIs) by user-submitted data or all available pre-loaded degradome data. Besides, the server can construct degradome-based miRNA regulatory networks (MRNs) based on the validated MTIs to help study the functions and relations among miRNAs and target mRNAs. WPMIAS is also suitable for other small RNAs (sRNAs), such as 21-nt phased siRNAs (phasiRNAs) and natural antisense siRNAs (nat-siRNAs), which direct cleavage of target mRNAs. Currently, WPMIAS supports 64 plant species with ∼200 cDNA libraries and 274 pre-loaded plant degradome datasets. The user can identify all validated MTIs by analyzing all degradome data at a time and understand when and where MTIs take place and their cleavage levels. With the data obtained from WPMIAS, the user can build a plant miRNA-target map, where it is convenient to find interesting research ideas on miRNAs. In summary, WPMIAS is able to support a comprehensive web-based plant miRNA-target analysis and expected to greatly promote future research on plant miRNAs. AVAILABILITY: It can be freely accessed at https://cbi.njau.edu.cn/WPMIAS/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Construction and analysis of degradome-dependent microRNA regulatory networks in soybean.

BACKGROUND: Usually the microRNA (miRNA)-mediated gene regulatory network (GRN) is constructed from the investigation of miRNA expression profiling and target predictions. However, the higher/lower expression level of miRNAs does not always indicate the higher/lower level of cleavages and such analysis, thus, sometimes ignores the crucial cleavage events. In the present work, the degradome sequencing data were employed to construct the complete miRNA-mediated gene regulatory network in soybean, unlike the traditional approach starting with small RNA sequencing data. RESULTS: We constructed the root-, cotyledon-, leaf- and seed-specific miRNA regulatory networks with the degradome sequencing data and the forthcoming verification of miRNA profiling analysis. As a result, we identified 205 conserved miRNA-target interactions (MTIs) involved with 6 conserved gma-miRNA families and 365 tissue-specific MTIs containing 24 root-specific, 45 leaf-specific, 63 cotyledon-specific and 225 seed-specific MTIs. We found a total of 156 miRNAs in tissue-specific MTIs including 18 tissue-specific miRNAs, however, only 3 miRNAs have consistent tissue-specific expression. Our study showed the degradome-dependent miRNA regulatory networks (DDNs) in four soybean tissues and explored their conservations and specificities. CONCLUSIONS: The construction of DDNs may provide the complete miRNA-Target interactions in certain plant tissues, leading to the identification of the conserved and tissue-specific MTIs and sub-networks. Our work provides a basis for further investigation of the roles and mechanisms of miRNA-mediated regulation of tissue-specific growth and development in soybean.

DPMIND: degradome-based plant miRNA-target interaction and network database.

Motivation: MicroRNAs (miRNAs) play essential roles in plant growth, development and stress responses through post-transcriptionally regulating the expression levels of their target mRNAs. Although some tools and databases were developed for predicting the relationships between miRNAs and their targets (miR-Tar), most of them were dependent on computational methods without experimental validations. With the development of degradome sequencing techniques, researchers can identify potential interactions based on degradome sequencing data. The validations with specific degradome data are useful to identify the miR-Tar interactions (MTIs) occurring in/under some specific tissues or treatments. Degradome-based plant miRNA-target interaction and network database (DPMIND) collected almost all available plant degradome data and built a retrieval and analysis platform of miRNA-target interactions and miRNA regulatory networks (MRNs). Results: DPMIND contains the recently updated 3794 miRNAs and 28 666 verified MTIs with 69 degradomes from 10 plant species. Not only the verified MTIs but also the degradome-based MRNs can be retrieved from DPMIND. Users can search for the verified MTIs and build degradome-based MRNs for the specific miRNAs or targets. DPMIND can build the MRNs based on all degradomes or specific degradomes, which helps to identify all possible connections among specific miR-Tar and compare the miRNA-mediated networks among various tissues or treatments. It can also build the networks mediated by all known miRNAs based on specific degradomes. Furthermore, DPMIND can be used to study the conservation and specificity of MTIs and sub-networks across different plant tissues or species. Availability and implementation: http://202.195.246.60/DPMIND/. Contact: huangji@njau.edu.cn. Supplementary information: Supplementary data are available at Bioinformatics online.

RNA Structural Dynamics Modulate EGFR-TKI Resistance Through Controlling YRDC Translation in NSCLC Cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control ratio of non-small cell lung cancer (NSCLC). Rapidly acquired resistance to EGFR-TKI is a major hurdle in successful treatment. However, the mechanisms that control the resistance of EGFR-TKI remain largely unknown. RNA structures have widespread and crucial functions in many biological regulations; however, the functions of RNA structures in regulating cancer drug resistance remain unclear. Here, the psoralen analysis of RNA interactions and structures (PARIS) method is used to establish the higher-order RNA structure maps of EGFR-TKI-resistant and -sensitive cells of NSCLC. Our results show that RNA structural regions are enriched in untranslated regions (UTRs) and correlate with translation efficiency (TE). Moreover, yrdC N6-threonylcarbamoyltransferase domain containing (YRDC) promotes resistance to EGFR-TKI. RNA structure formation in YRDC 3' UTR suppresses embryonic lethal abnormal vision-like 1 (ELAVL1) binding, leading to EGFR-TKI sensitivity by impairing YRDC translation. A potential cancer therapy strategy is provided using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism through which the RNA structure switch modulates EGFR-TKI resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner.

A Conserved NRDE-2/MTR-4 Complex Mediates Nuclear RNAi in Caenorhabditis elegans.

Small regulatory RNAs, such as small interfering RNAs (siRNAs) and PIWI-interacting RNAs, regulate splicing, transcription, and genome integrity in many eukaryotes. In Caenorhabditis elegans, siRNAs bind nuclear Argonautes (AGOs), which interact with homologous premessenger RNAs to recruit downstream silencing effectors, such as NRDE-2, to direct cotranscriptional gene silencing [or nuclear RNA interference (RNAi)]. To further our understanding of the mechanism of nuclear RNAi, we conducted immunoprecipitation-mass spectrometry on C. elegans NRDE-2 The major NRDE-2 interacting protein identified was the RNA helicase MTR-4 Co-immunoprecipitation analyses confirmed a physical association between NRDE-2 and MTR-4 MTR-4 colocalizes with NRDE-2 within the nuclei of most/all C. elegans somatic and germline cells. MTR-4 is required for nuclear RNAi, and interestingly, MTR-4 is recruited to premessenger RNAs undergoing nuclear RNAi via a process requiring nuclear siRNAs, the nuclear AGO HRDE-1, and NRDE-2, indicating that MTR-4 is a component of the C. elegans nuclear RNAi machinery. Finally, we confirm previous reports showing that human (Hs)NRDE2 and HsMTR4 also physically interact. Our data show that the NRDE-2/MTR-4 interactions are evolutionarily conserved, and that, in C. elegans, the NRDE-2/MTR-4 complex contributes to siRNA-directed cotranscriptional gene silencing.