Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called Vetting & Analysis of RNA for in situ Hybridization probes (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.

Small RNAs play important roles during plant development by regulating transcript levels of target mRNAs, maintaining genome integrity, and reinforcing DNA methylation. Dicer-like 5 (Dcl5) is proposed to be responsible for precise slicing in many monocots to generate diverse 24-nt phased, secondary small interfering RNAs (phasiRNAs), which are exceptionally abundant in meiotic anthers of diverse flowering plants. The importance and functions of these phasiRNAs remain unclear. Here, we characterized several mutants of dcl5, including alleles generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and a transposon-disrupted allele. We report that dcl5 mutants have few or no 24-nt phasiRNAs, develop short anthers with defective tapetal cells, and exhibit temperature-sensitive male fertility. We propose that DCL5 and 24-nt phasiRNAs are critical for fertility under growth regimes for optimal yield.

Maize anthers, the male reproductive floral organs, express two classes of phased small-interfering RNAs (phasiRNAs). PhasiRNA precursors are transcribed by RNA polymerase II and map to low-copy, intergenic regions similar to PIWI-interacting RNAs (piRNAs) in mammalian testis. From 10 sequential cohorts of staged maize anthers plus mature pollen we find that 21-nt phased siRNAs from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, whereas 24-nt phasiRNAs from 176 loci coordinately accumulate during meiosis and persist as anther somatic cells mature and haploid gametophytes differentiate into pollen. Male-sterile ocl4 anthers defective in epidermal signaling lack 21-nt phasiRNAs. Male-sterile mutants with subepidermal defects--mac1 (excess meiocytes), ms23 (defective pretapetal cells), and msca1 (no normal soma or meiocytes)--lack 24-nt phasiRNAs. ameiotic1 mutants (normal soma, no meiosis) accumulate both 21-nt and 24-nt phasiRNAs, ruling out meiotic cells as a source or regulator of phasiRNA biogenesis. By in situ hybridization, miR2118 triggers of 21-nt phasiRNA biogenesis localize to epidermis; however, 21-PHAS precursors and 21-nt phasiRNAs are abundant subepidermally. The miR2275 trigger, 24-PHAS precursors, and 24-nt phasiRNAs all accumulate preferentially in tapetum and meiocytes. Therefore, each phasiRNA type exhibits independent spatiotemporal regulation with 21-nt premeiotic phasiRNAs dependent on epidermal and 24-nt meiotic phasiRNAs dependent on tapetal cell differentiation. Maize phasiRNAs and mammalian piRNAs illustrate putative convergent evolution of small RNAs in male reproduction.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence Spinach, a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.

MOTIVATION: Tumor-specific antigen (TSA) identification in human cancer predicts response to immunotherapy and provides targets for cancer vaccine and adoptive T-cell therapies with curative potential, and TSAs that are highly expressed at the RNA level are more likely to be presented on major histocompatibility complex (MHC)-I. Direct measurements of the RNA expression of peptides would allow for generalized prediction of TSAs. Human leukocyte antigen (HLA)-I genotypes were predicted with seq2HLA. RNA sequencing (RNAseq) fastq files were translated into all possible peptides of length 8-11, and peptides with high and low expressions in the tumor and control samples, respectively, were tested for their MHC-I binding potential with netMHCpan-4.0. RESULTS: A novel pipeline for TSA prediction from RNAseq was used to predict all possible unique peptides size 8-11 on previously published murine and human lung and lymphoma tumors and validated on matched tumor and control lung adenocarcinoma (LUAD) samples. We show that neoantigens predicted by exomeSeq are typically poorly expressed at the RNA level, and a fraction is expressed in matched normal samples. TSAs presented in the proteomics data have higher RNA abundance and lower MHC-I binding percentile, and these attributes are used to discover high confidence TSAs within the validation cohort. Finally, a subset of these high confidence TSAs is expressed in a majority of LUAD tumors and represents attractive vaccine targets. AVAILABILITY AND IMPLEMENTATION: The datasets were derived from sources in the public domain as follows: TSAFinder is open-source software written in python and R. It is licensed under CC-BY-NC-SA and can be downloaded at https://github.com/RNAseqTSA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fast-spiking (FS) neurons can fire action potentials (APs) up to 1,000 Hz and play key roles in vital functions such as sound location, motor coordination, and cognition. Here we report that the concerted actions of Kv3 voltage-gated K⁺ (Kv) and Na⁺ (Nav) channels are sufficient and necessary for inducing and maintaining FS. Voltage-clamp analysis revealed a robust correlation between the Kv3/Nav current ratio and FS. Expressing Kv3 channels alone could convert ∼30%-60% slow-spiking (SS) neurons to FS in culture. In contrast, co-expression of either Nav1.2 or Nav1.6 together with Kv3.1 or Kv3.3, but not alone or with Kv1.2, converted SS to FS with 100% efficiency. Furthermore, RNA-sequencing-based genome-wide analysis revealed that the Kv3/Nav ratio and Kv3 expression levels strongly correlated with the maximal AP frequencies. Therefore, FS is established by the properly balanced activities of Kv3 and Nav channels and could be further fine-tuned by channel biophysical features and localization patterns.