A bias-reducing strategy in profiling small RNAs using Solexa

Small RNAs (smRNAs) encompass several different classes of short noncoding RNAs. Progress in smRNA research and applications has coincided with the advance of techniques to detect them. Next-generation sequencing technologies are becoming the preferred smRNA profiling method because of their high-throughput capacity and digitized results. In our small RNA profiling study using Solexa, we observed serious biases introduced by the 5' adaptors in small RNA species coverage and abundance; therefore, the results cannot reveal the accurate composition of the small RNAome. We found that the profiling results can be significantly optimized by using an index pool of 64 customized 5' adaptors. This pool of 64 adaptors can be further reduced to four smaller index pools, each containing 16 adaptors, to minimize profiling bias and facilitate multiplexing. It is plausible that this type of bias exists in other deep-sequencing technologies, and adaptor pooling could be an easy work-around solution to reveal the "true" small RNAome.     

Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs

Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNA^Gln is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.     

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca)

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.     

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum

Background

Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms.

Results

Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions.

Conclusions

P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-698) contains supplementary material, which is available to authorized users.     

Identification of small non-coding RNAs in the planarian Dugesia japonica via deep sequencing

Freshwater planarian flatworm possesses an extraordinary ability to regenerate lost body parts after amputation; it is perfect organism model in regeneration and stem cell biology. Recently, small RNAs have been an increasing concern and studied in many aspects, including regeneration and stem cell biology, among others. In the current study, the large-scale cloning and sequencing of sRNAs from the intact and regenerative planarian Dugesia japonica are reported. Sequence analysis shows that sRNAs between 18nt and 40nt are mainly microRNAs and piRNAs. In addition, 209 conserved miRNAs and 12 novel miRNAs are identified. Especially, a better screening target method, negative-correlation relationship of miRNAs and mRNA, is adopted to improve target prediction accuracy. Similar to miRNAs, a diverse population of piRNAs and changes in the two samples are also listed. The present study is the first to report on the important role of sRNAs during planarian Dugesia japonica regeneration.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.     

A particular set of small non-coding RNAs is bound to the distinctive Argonaute protein of Trypanosoma cruzi: Insights from RNA-interference deficient organisms

The study of small RNAs and Argonaute proteins in eukaryotes that are deficient in functional RNA interference could provide insights into novel functions of small RNAs. In this study we describe small non-coding RNAs bound to a distinctive Argonaute protein of Trypanosoma cruzi, TcPIWI-tryp. Co-immunoprecipitation of TcPIWI-tryp followed by deep sequencing of isolated RNA identified abundant small RNAs derived from rRNAs and tRNAs. The small RNA repertoire differed from that of the canonical Argonaute in organisms with functional RNA interference, which could indicate novel biological functions for TcPIWI-tryp in T. cruzi and other members of the trypanosomatid clade.     

链球菌非编码小RNA研究进展

非编码小RNA(Small non-coding RNA,sRNA)是一种存在于原核和真核生物中的新型调控RNA,长度约为40～500个核苷酸。作为一类关键的调控因子,sRNA通过与靶mRNA或蛋白质结合来调控细胞内的基因表达。大部分细菌sRNA在大肠杆菌等革兰氏阴性菌中被发现并研究,但近十年来越来越多的sRNA在革兰氏阳性菌中被逐步发现。作为一类革兰氏阳性菌,链球菌属中sRNA目前研究主要集中在毒力调节,鲜有其他调控的报道。本文总结了链球菌中sRNA的最新进展,并介绍其主要功能和机理,以期为细菌sRNA研究提供借鉴。     

细菌sRNA对环境胁迫的响应机制

细菌小RNA (Small RNAs,sRNAs)是一类长度大约在40?400个核酸之间,不编码蛋白质的RNA,在细菌适应环境方面起重要的调节作用。当环境中温度、营养、外膜蛋白、pH、铁等条件改变时,sRNA常常通过连接双组分信号转导系统和调节蛋白,来传递压力信号并调节应激响应,其作用方式一般是通过碱基互补配对的方式与靶mRNA结合,从而调控靶mRNA的翻译和稳定性;或直接与靶标蛋白质结合,调节靶标蛋白质的生物活性。本文总结了细菌在多种环境压力下,sRNA的调控响应机制。     

Genome-wide analysis of long non-coding RNAs in Pichia pastoris during stress by RNA sequencing

Long non-coding RNAs play significant roles in many biological processes. The roles of lncRNAs in Pichia pastoris remain unclear. In this work, we focused on the identification of lncRNAs in P. pastoris and exploration of their potential roles in stress response to PLA₂ overexpression and methanol induction. By strand specific RNA sequencing, 208 novel long non-coding RNAs were identified and analyzed. Bioinformatic analysis showed potential trans-target genes and cis-regulated genes of 39 differential lncRNAs. Functional annotation and sequence motif analysis indicated that lncRNAs participate in pathways related to methanol degradation and production of the recombinant protein. The differential expression of lncRNAs was validated by qRT-PCR. Lastly, the potential functions of three lncRNAs were evaluated by knockdown of their expression and analysis of the expression levels of target genes. Our study identifies novel lncRNAs in P. pastoris induced during use as a bioreactor, facilitating future functional research.