DANSR: A Tool for the Detection of Annotated and Novel Small RNAs

Existing small noncoding RNA analysis tools are optimized for processing short sequencing reads (17–35 nucleotides) to monitor microRNA expression. However, these strategies under-represent many biologically relevant classes of small noncoding RNAs in the 36–200 nucleotides length range (tRNAs, snoRNAs, etc.). To address this, we developed DANSR, a tool for the detection of annotated and novel small RNAs using sequencing reads with variable lengths (ranging from 17–200 nt). While DANSR is broadly applicable to any small RNA dataset, we applied it to a cohort of matched normal, primary, and distant metastatic colorectal cancer specimens to demonstrate its ability to quantify annotated small RNAs, discover novel genes, and calculate differential expression. DANSR is available as an open source tool.

Spatiotemporal expression profile of novel and known small RNAs throughout rice plant development focussing on seed tissues

Background Small RNAs (sRNAs) regulate numerous plant processes directly related to yield, such as disease resistance and plant growth. To exploit this yield-regulating potential of sRNAs, the sRNA profile of one of the world’s most important staple crops – rice – was investigated throughout plant development using next-generation sequencing. Results Root and leaves were investigated at both the vegetative and generative phase, and early-life sRNA expression was characterized in the embryo and endosperm. This led to the identification of 49,505 novel sRNAs and 5581 tRNA-derived sRNAs (tsRNAs). In all tissues, 24 nt small interfering RNAs (siRNAs) were highly expressed and associated with euchromatic, but not heterochromatic transposable elements. Twenty-one nt siRNAs deriving from genic regions in the endosperm were exceptionally highly expressed, mimicking previously reported expression levels of 24 nt siRNAs in younger endosperm samples. In rice embryos, sRNA content was highly diverse while tsRNAs were underrepresented, possibly due to snoRNA activity. Publicly available mRNA expression and DNA methylation profiles were used to identify putative siRNA targets in embryo and endosperm. These include multiple genes related to the plant hormones gibberellic acid and ethylene, and to seed phytoalexin and iron content. Conclusions This work introduces multiple sRNAs as potential regulators of rice yield and quality, identifying them as possible targets for the continuous search to optimize rice production.

Weight Change Alters the Small RNA Profile of Urinary Extracellular Vesicles in Obesity

Introduction: Various kidney diseases reportedly show different urinary extracellular vesicles (EVs) RNA profiles. Although obesity is one of the main causes of chronic kidney disease, the expression pattern of urinary EVs RNA in obesity is uncertain. Our aim was to sequence the small RNA profiles of urinary EVs in obese patients before and after weight reduction and compared them to those of healthy volunteers (HVs). Methods: We recruited age-sex matched obese patients and HVs. The small RNA profiles of urinary EVs were analyzed using RNA sequencing. To evaluate the effect of weight reduction, small RNA profiles of urinary EVs 6 months after bariatric surgery were also analyzed. Results: The proportion of urinary EVs transfer RNA and microRNA of obese patients differed from that of HVs. Obese patients showed differential expression of 1343 small RNAs in urinary EVs compared to HVs (|fold change| ≥ 2 and p value < 0.05). Among those, 61 small RNAs were upregulated in obese patients and downregulated after weight reduction, whereas 167 small RNAs were downregulated in obese patients and upregulated after weight reduction. RNA sequencing revealed the correlation between the specific urinary EVs small RNAs and clinical parameters including body weight, low-density lipoprotein cholesterol, triglyceride, high-density lipoprotein cholesterol, serum glucose, estimated glomerular filtration rate, and albuminuria. Conclusion: Obese patients showed distinct urinary EVs small RNA profiles compared to HVs. Weight reduction altered urinary EVs small RNA profiles in obese patients.

Small RNAs Participate in Plant–Virus Interaction and Their Application in Plant Viral Defense

Small RNAs are significant regulators of gene expression, which play multiple roles in plant development, growth, reproductive and stress response. It is generally believed that the regulation of plants’ endogenous genes by small RNAs has evolved from a cellular defense mechanism for RNA viruses and transposons. Most small RNAs have well-established roles in the defense response, such as viral response. During viral infection, plant endogenous small RNAs can direct virus resistance by regulating the gene expression in the host defense pathway, while the small RNAs derived from viruses are the core of the conserved and effective RNAi resistance mechanism. As a counter strategy, viruses evolve suppressors of the RNAi pathway to disrupt host plant silencing against viruses. Currently, several studies have been published elucidating the mechanisms by which small RNAs regulate viral defense in different crops. This paper reviews the distinct pathways of small RNAs biogenesis and the molecular mechanisms of small RNAs mediating antiviral immunity in plants, as well as summarizes the coping strategies used by viruses to override this immune response. Finally, we discuss the current development state of the new applications in virus defense based on small RNA silencing.

Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus

RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhanching RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.

Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA Induced Silencing Complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to down regulation of the corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3′ supplemental regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

tRNA-derived fragments from wheat are potentially involved in susceptibility to Fusarium head blight

Background Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating fungal disease of wheat. The mechanism underlying F. graminearum-wheat interaction remains largely unknown. tRNA-derived fragments (tRFs) are RNase-dependent small RNAs derived from tRNAs, and they have not been reported in wheat yet, and whether tRFs are involved in wheat-F. graminearum interactions remains unknown. Results Herein, small RNAs from the spikelets inoculated with F. graminearum and mock from an FHB-susceptible variety Chinese Spring (CS) and an FHB-resistant variety Sumai3 (SM) were sequenced respectively. A total of 1249 putative tRFs were identified, in which 15 tRFs was CS-specific and 12 SM-specific. Compared with mock inoculation, 39 tRFs were significantly up-regulated across both wheat varieties after F. graminearum challenge and only nine tRFs were significantly down-regulated. tRFGlu, tRFLys and tRFThr were dramatically induced by F. graminearum infection, with significantly higher fold changes in CS than those in SM. The expression patterns of the three highly induced tRFs were further validated by stem-loop qRT-PCR. The accumulation of tRFs were closely related to ribonucleases T2 family members, which were induced by F. graminearum challenge. The tRFs’ targets in host were predicted and were validated by RNA sequencing. Conclusion Integrative analysis of the differentially expressed tRFs and their candidate targets indicated that tRFGlu, tRFLys and tRFThr might negatively regulate wheat resistance to FHB. Our results unvealed the potential roles of tRFs in wheat-F. graminearum interactions.

Differential Expression Profiles and Potential Intergenerational Functions of tRNA-Derived Small RNAs in Mice After Cadmium Exposure

Cadmium (Cd) is a toxic heavy metal and ubiquitous environmental endocrine disruptor. Previous studies on Cd-induced damage to male fertility mainly focus on the structure and function of testis, including cytoskeleton, blood-testis barrier, and steroidogenesis. Nevertheless, to date, no studies have investigated the effects of Cd exposure on sperm epigenetic inheritance and intergenerational inheritance. In our study, we systematically revealed the changes in sperm tRNA-derived small RNAs (tsRNA) profiles and found that 14 tsRNAs (9 up-regulated and 5 down-regulated) were significantly altered after Cd exposure. Bioinformatics of tsRNA-mRNA-pathway interactions revealed that the altered biological functions mainly were related to ion transmembrane transport, lipid metabolism and cell membrane system. In addition, we focused on two stages of early embryo development and selected two organs to study the impact of these changes on cell membrane system, especially mitochondrion and lysosome, two typical membrane-enclosed organelles. Surprisingly, we found that the content of mitochondrion was significantly decreased in 2-cell stage, whereas remarkably increased in the morula stage. The contents of mitochondrion and lysosome were increased in the testes of 6-day-old offspring and livers of adult offspring, whereas remarkably decreased in the testes of adult offspring. This provides a possible basis to further explore the effects of paternal Cd exposure on offspring health.

Pilot study of changes in the level of piRNA in plasma and serum in women at different stages of physiological pregnancy

Aim. To study changes in the level of piRNA in plasma and serum of pregnant women at different stages of gestation.Material and Methods. A total of 42 samples of plasma and blood serum were obtained from seven women with physiological singleton pregnancy without obstetric and gynecological pathology. The study was carried out at three time points corresponding to 8–13, 18–25, and 30–35 weeks of pregnancy, respectively. To assess the spectrum and levels of piRNA by the NGS method, whole genome sequencing of small RNAs was carried out. Sequencing data analysis was performed using the GeneGlobe Data Analysis Center web application. Differential expression was assessed using the DESeq2 R package.Results and Discussion. The piRNA contents among all small RNAs were 2.29%, 2.61%, and 4.16% in plasma and 7.29%, 7.02%, and 10.82% in serum during the first, second, and third trimesters, respectively. The contents of the following piRNAs increased in blood plasma from the first to the third trimester: piR 000765, piR 020326, piR 019825, piR 020497, piR 015026, piR 001312, and piR 017716. The study showed that the levels of piR 000765, piR 020326, piR 019825, piR 015026, piR 020497, piR 001312, piR 017716, and piR 004153 were significantly higher in serum compared with the corresponding values in plasma whereas the content of only one molecule, piR 018849, was higher in plasma.Conclusion. This pilot work created a basis for understanding the processes of piRNA expression in plasma and serum of pregnant women and can become the foundation for the search for biomarkers of various complications in pregnancy.