Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs

Small virus-derived interfering RNAs (viRNAs) play an important role in antiviral defence in plants, insects and nematodes by triggering the RNA interference (RNAi) pathway. The role of RNAi as an antiviral defence mechanism in mammalian cells has been obscure due to the lack of viRNA detection. Although viRNAs from different mammalian viruses have recently been identified, their functions and possible impact on viral replication remain unknown. To identify viRNAs derived from HIV-1, we used the extremely sensitive SOLiD(TM) 3 Plus System to analyse viRNA accumulation in HIV-1-infected T lymphocytes. We detected numerous small RNAs that correspond to the HIV-1 RNA genome. The majority of these sequences have a positive polarity (98.1%) and could be derived from miRNAs encoded by structured segments of the HIV-1 RNA genome (vmiRNAs). A small portion of the viRNAs is of negative polarity and most of them are encoded within the 3'-UTR, which may represent viral siRNAs (vsiRNAs). The identified vsiRNAs can potently repress HIV-1 production, whereas suppression of the vsiRNAs by antagomirs stimulate virus production. These results suggest that HIV-1 triggers the production of vsiRNAs and vmiRNAs to modulate cellular and/or viral gene expression.     

The modularity of biological regulatory networks

A useful approach to complex regulatory networks consists of modeling their elements and interactions by Boolean equations. In this context, feedback circuits (i.e. circular sequences of interactions) have been shown to play key dynamical roles: whereas positive circuits are able to generate multistationarity, negative circuits may generate oscillatory behavior. In this paper, we principally focus on the case of gene networks. These are represented by fully connected Boolean networks where each element interacts with all elements including itself. Flexibility in network design is introduced by the use of Boolean parameters, one associated with each interaction or group of interactions affecting a given element. Within this formalism, a feedback circuit will generate its typical dynamical behavior (i.e. multistationarity or oscillations) only for appropriate values of some of the logical parameters. Whenever it does, we say that the circuit is 'functional'. More interestingly, this formalism allows the computation of the constraints on the logical parameters to have any feedback circuit functional in a network. Using this methodology, we found that the fraction of the total number of consistent combinations of parameter values that make a circuit functional decreases geometrically with the circuit length. From a biological point of view, this suggests that regulatory networks could be decomposed into small and relatively independent feedback circuits or 'regulatory modules'.     

Large-scale analysis of small RNAs derived from traditional Chinese herbs in human tissues

Plant-derived microRNAs have recently been reported to function in human blood and tissues. Controversy was immediately raised due to possible contamination and the lack of large sample sizes. Here, we report thousands of unique small RNAs derived from traditional Chinese medicine(TCM) herbs found in human blood cells and mouse lung tissues using a large-scale analysis.We extracted small RNAs from decoctions of 10 TCM plants(Ban Zhi Lian, Chai Hu, Chuan Xin Lian, Di Ding Zi Jin, Huang Qin, Jin Yin Hua, Lian Qiao, Pu Gong Ying, Xia Ku Cao, and Yu Xing Cao) and obtained millions of RNA sequences from each herb. We also obtained RNA-Seq data from the blood cells of humans who consumed herbal decoctions and from the lung tissues of mice administered RNAs from herbal decoctions via oral gavage. We identified thousands of unique small RNA sequences in human blood cells and mouse lung tissues. Some of these identified small RNAs from Chuan Xin Lian and Hong Jing Tian could be mapped to the genomes of the herbs, confirming their TCM plant origin. Small RNAs derived from herbs regulate mammalian gene expression in a sequence-specific manner, and thus are a superior novel class of herbal drug components that hold great potential as oral gene-targeted therapeutics, highlighting the important role of herbgenomics in their development.     

Virus, phage, transposon and their regulatory small non-coding RNAs

Many reports have been accumulated describing not a few microRNAs (miRNAs) in eukaryotes target viral genomes, whereas a number of viruses also encode miRNA genes. These small RNAs play important roles on viral infection and their replication. In germ cells, another small RNA, piRNA is reported to repress endogenous transposons. Furthermore, CRISPR RNA target virus/phage genomes in both archaea and bacteria. Therefore, small RNA is deeply involved in a broad range of biological defense systems. This system may be applied not only to control replication of viruses or phages but also provide implication on regulating the growth of microorganisms including pathogenic bacteria.     

Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing

Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.     

Assessing the gene regulatory properties of Argonaute-bound small RNAs of diverse genomic origin

High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles.     

Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.     

A genome-wide identification analysis of small regulatory RNAs in Mycobacterium tuberculosis by RNA-Seq and conservation analysis

We propose a new method for smallRNAs (sRNAs) identification. First we build an effective target genome (ETG) by means of a strand-specific procedure. Then we propose a new bioinformatic pipeline based mainly on the combination of two types of information: the first provides an expression map based on RNA-seq data (Reads Map) and the second applies principles of comparative genomics leading to a Conservation Map. By superimposing these two maps, a robust method for the search of sRNAs is obtained. We apply this methodology to investigate sRNAs in Mycobacterium tuberculosis H37Rv. This bioinformatic procedure leads to a total list of 1948 candidate sRNAs. The size of the candidate list is strictly related to the aim of the study and to the technology used during the verification process. We provide performance measures of the algorithm in identifying annotated sRNAs reported in three recent published studies.     

Small regulatory RNAs in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is frequently associated with nosocomial infections, and can be life threatening in immunosuppressed, cancer and cystic fibrosis patients. Virulence in P. aeruginosa is combinatorial, and results from the activation of several genetic programs that regulate motility, attachment to the host epithelium as well as the synthesis of exotoxins. The pathogen has a high survival capacity in the host owing to its metabolic versatility, nutrient scavenging and resistance against both, antibiotics and immune defenses. Adaptive responses to various environmental stresses and stimuli are often regulated by small regulatory RNAs (sRNA). In this review, we summarize the current knowledge on the regulation and function of P. aeruginosa sRNAs that titrate regulatory proteins, base-pair with target mRNAs, and which are derived from CRISPR elements.     

Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs

Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.