A sensitive non-radioactive northern blot method to detect small RNAs

The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (∼15–40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to ∼1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data

Transfer RNA-derived small RNAs in the cancer transcriptome

The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis.These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such disruption takes place. RNA silencing is a term used to describe the effects of post-transcriptional gene silencing mediated by a diverse set of small RNA molecules. Small RNAs are crucial for regulating gene expression and microguarding genome integrity.RNA silencing studies predominantly focus on small RNAs such as microRNAs, short-interfering RNAs and piwi-interacting RNAs. We describe an emerging renewal of inter-est in a‘larger’small RNA, the transfer RNA (tRNA).Precisely generated tRNA-derived small RNAs, named tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been reported to be abundant with dysregulation associated with cancer. Transfection of tiRNAs inhibits protein translation by displacing eukaryotic initiation factors from messenger RNA (mRNA) and inaugurating stress granule formation.Knockdown of an overexpressed tRF inhibits cancer cell proliferation. Recovery of lacking tRFs prevents cancer metastasis. The dual oncogenic and tumour-suppressive role is typical of functional small RNAs. We review recent reports on tiRNA and tRF discovery and biogenesis, identification and analysis from next-generation sequencing data and a mechanistic animal study to demonstrate their physiological role in cancer biology. We propose tRNA-derived small RNA-mediated RNA silencing is an innate defence mechanism to prevent oncogenic translation. We expect that cancer cells are percipient to their ablated control of transcription and attempt to prevent loss of genome control through RNA silencing

Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

Expansion of the miRNA Pathway in the Hemipteran Insect Acyrthosiphon pisum

The pathways that allow short noncoding RNAs such as the microRNAs (miRNAs) to mediate gene regulation and control critical cellular and developmental processes involve a limited number of key protein components. These proteins are the Dicer-like RNases, double-stranded RNA (dsRNA)-binding proteins, and the Argonaute (AGO) proteins that process stem-loop hairpin transcripts of endogenous genes to generate miRNAs or long dsRNA precursors (either exogenous or endogenous). Comparative genomics studies of metazoans have shown the pathways to be highly conserved overall; the major difference observed is that the vertebrate pathways overlap in sharing a single Dicer (DCR) and AGO proteins, whereas those of insects appear to be parallel, with distinct Dicers and AGOs required for each pathway. The genome of the pea aphid is the first available for a hemipteran insect and discloses an unexpected expansion of the miRNA pathway. It has two copies of the miRNA-specific dicr-1 and ago1 genes and four copies of pasha a cofactor of drosha involved in miRNA biosynthesis. For three of these expansions, we showed that one copy of the genes diverged rapidly and in one case (ago1b) shows signs of positive selection. These expansions occurred concomitantly within a brief evolutionary period. The pea aphid, which reproduces by viviparous parthenogenesis, is able to produce several adapted phenotypes from one single genotype. We show by reverse transcriptase-polymerase chain reaction that all the duplicated copies of the miRNA machinery genes are expressed in the different morphs. Investigating the function of these novel genes offers an exciting new challenge in aphid biology

A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1(-) M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi

An “In-Depth” Description of the Small Non-coding RNA Population of Schistosoma japonicum Schistosomulum

Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, which afflicts more than 200 million people yearly in tropical regions of South America, Asia and Africa. A promising approach to the control of this and many other diseases involves the application of our understanding of small non-coding RNA function to the design of safe and effective means of treatment. In a previous study, we identified five conserved miRNAs from the adult stage of Schistosoma japonicum. Here, we applied Illumina Solexa high-throughput sequencing methods (deep sequencing) to investigate the small RNAs expressed in S. japonicum schistosomulum (3 weeks post-infection). This has allowed us to examine over four million sequence reads including both frequently and infrequently represented members of the RNA population. Thus we have identified 20 conserved miRNA families that have orthologs in well-studied model organisms and 16 miRNA that appear to be specific to Schistosoma. We have also observed minor amounts of heterogeneity in both 3′ and 5′ terminal positions of some miRNA as well as RNA fragments resulting from the processing of miRNA precursor. An investigation of the genomic arrangement of the 36 identified miRNA revealed that seven were tightly linked in two clusters. We also identified members of the small RNA population whose structure indicates that they are part of an endogenously derived RNA silencing pathway, as evidenced by their extensive complementarities with retrotransposon and retrovirus-related Pol polyprotein from transposon

Transposable-Element Associated Small RNAs in Bombyx mori Genome

Small RNAs are a group of regulatory RNA molecules that control gene expression at transcriptional or post-transcriptional levels among eukaryotes. The silkworm, Bombyx mori L., genome harbors abundant repetitive sequences derived from families of retrotransposons and transposons, which together constitute almost half of the genome space and provide ample resource for biogenesis of the three major small RNA families. We systematically discovered transposable-element (TE)-associated small RNAs in B. mori genome based on a deep RNA-sequencing strategy and the effort yielded 182, 788 and 4,990 TE-associated small RNAs in the miRNA, siRNA and piRNA species, respectively. Our analysis suggested that the three small RNA species preferentially associate with different TEs to create sequence and functional diversity, and we also show evidence that a Bombyx non-LTR retrotransposon, bm1645, alone contributes to the generation of TE-associated small RNAs in a very significant way. The fact that bm1645-associated small RNAs partially overlap with each other implies a possibility that this element may be modulated by different mechanisms to generate different products with diverse functions. Taken together, these discoveries expand the small RNA pool in B. mori genome and lead to new knowledge on the diversity and functional significance of TE-associated small RNAs

Suppression of MMP-2 Attenuates TNF-α Induced NF-κB Activation and Leads to JNK Mediated Cell Death in Glioma

BACKGROUND: Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. METHODOLOGY/PRINCIPAL FINDINGS: MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. CONCLUSION/SIGNIFICANCE: In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo

Profiles of Small Non-Coding RNAs in Schistosoma japonicum during Development

Schistosomiasis, a debilitating disease, caused by agents of the genus Schistosoma afflicts more than 200 million people worldwide. Schistosomes could serve as an interesting model to explore gene regulation due to its evolutional position, complex life cycle and sexual dimorphism. We previously indicated that sncRNA profile in the parasite S. japonicum was developmentally regulated in hepatic and adult stages. In this study, we systematically investigated mircoRNA (miRNA) and endogenous siRNA (endo-siRNA) profile in this parasite in more detailed developmental stages (cercariae, lung-stage schistosomula, separated adult worms, and liver tissue-trapped eggs) using high-throughput RNA sequencing technology. We observed that the ratio of miRNAs to endo-siRNAs was dynamically changed throughout different developmental stages of the parasite. MiRNAs were expressed dominantly in cercariae, while endo-siRNAs accumulated in adult female worms and hepatic eggs. We demonstrated that miRNAs were mostly derived from intergenic regions whereas siRNAs were mostly derived from transposable elements. We also annotated miRNAs and siRNAs with stage- and gender- biased expression. Our findings would facilitate to understand the gene regulation mechanism of this parasite and discover novel targets for anti-parasite drugs