Impact of Small RNA Sponges on Regulatory RNA Networks in Bacteria.

There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the gltIJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the gltIJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources.

In the recent years, a huge number of human transcripts have been found in the human genome that do not encode for proteins, which have been named non-coding RNAs (npcRNAs) containing secondary structures or short regions highly conserved within mammalian sequences. Long RNAs (antisense RNA, structured RNA, and long interspersed ncRNAs) and small RNAs (miRNAs, siRNAs, snoRNAs) have shown to exert many roles: functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, controlling protein ubiquitination and degradation, regulating epigenetic marks and affecting chromosome structure. One group of npcRNAs that is well-characterized, at the biochemical level, is represented by miRNAs. This group comprises a large class of small npcRNAs (~22-nucleotide RNAs) acting through base pairing to partially complementary sites in the 3’untranslated regions (3’UTR) of the targeted messenger RNA. Circular RNAs, competing endogenous RNAs (ceRNAs) acting as RNA sponges, natural antisense RNAs (NAT), enhancer RNAs (eRNAs), and RNA decoys are further expanding the wide array of functionalities exerted by ncRNAs. In the last case, as example, Growth Arrest Suppressor 5 (GAS5) forms a structured RNA tha is a decoy for the glucocorticoid receptor (GR), mimicking the DNA structure of the GR element (GRE). Long noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Many of the mechanisms that modify the 3' UTRs, or that affect differential splicing, make use of RNA regulation, antisense RNAs, and may involve RNP complexes. Many human ncRNAs have been characterized in terms of function or expression profiles. HOTAIR, described in detail in the review by Ge Shan, is a structured RNA that assemble several proteins to form an epigenetic regulation complex: it assembles Polycomb Repressive Complex (PRC) proteins and determines the silencing of specific genes. Terminal differentiation induced ncRNA (TINCR) destabilises ALU elements in mRNAs through the RNA binding protein Staufen 1 binding to polypurine tract. Thus, proteins involved in the functioning of ncRNAs are highly varied: RNA binding proteins, ribonucleoprotein complexes, alternative splicing proteins, alternative polyadenylation proteins, chromatin remodeling complexes, and gene activation and repression complexes (PRC) and enzymes positioning or eliminating histone marks. Then, it is clear that the changes determined in several diseases and in cancer are caused non only by mutated genes but also by epigenetic deregulation and by alternative spliced genes and alternative polyA tails that evade microRNA recognition. Concerning miRs, the varied presence of Argonaute family of proteins and the link with diseases are well detailed in more than one review in this special issue. In this thematic issue, Ge Shan's paper provides an overview of several examples in small and long RNAs; Yangchao Chen introduces the recent topic of circular RNAs; Charles Lawrie presents the deregulated pathways involving miRNAs in myeloma malignancies, while George Calin classifies leukemias according to epigenetic deregulation, oncomiRs and loss of antioncomiRs; Massimo Mallardo reviews the mechanism of infectivity in RNA viruses; Marek Sanak describes a unique microRNA differently expressed in neutrophils in healthy individuals and in Granulomatosis with Polyangiitis patients; finally two papers describe the circulating miRNAs protected by exosomes that are released by cancer cells and can be found in bodily fluids. Sonia Melo gives a detailed overview on circulating miRNAs, and Valeria Mezzolla proposes a method to group glioma and differentiate them from other malignancies based on the most representative circulating miRNAs. This field is a promising means to ensure novel approaches to treatment and more effective therapies exploiting ncRNAs.

Small regulatory RNAs (sRNAs) are involved in post-transcriptional control of important cellular processes and contribute to the success of a pathogen. Here, we use studies primarily selected from Salmonella enterica and Listeria monocytogenes to illustrate the current status of sRNA biology in important foodborne pathogens. We discuss how the regulatory activities of sRNAs can be affected by base pairing RNAs known as 'sponge RNAs', or by RNA-binding proteins, such as the newly discovered sRNA chaperone ProQ. Furthermore, we highlight recent findings for sRNAs with regulatory roles during infection, some of which are present in multiple copies, designated 'sibling sRNAs'. Importantly, knowledge on sRNA-mediated regulation can be exploited for biotechnological applications, such as in generating gene knockdowns to promote desired traits.

Bacterial Chaperone Protein Hfq Facilitates the Annealing of Sponge RNAs to Small Regulatory RNAs

LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance

miRNA regulatory variation in human evolution

Principles and Properties of Eukaryotic mRNPs

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.

The Elegance of the MicroRNAs: A Neuronal Perspective

Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

The Sponge RNAs of bacteria – How to find them and their role in regulating the post-transcriptional network

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

MicroRNAs (miRNAs) negatively regulate gene expression at the post-transcriptional level, primarily by base-pairing with the 3'-untranslated region (3'-UTR) of their target mRNAs. Many miRNAs are expressed in a tissue/organ-specific manner and are associated with an increasing number of cell proliferation, differentiation, and tissue development events. Cardiac muscle expresses distinct genes encoding structural proteins and a subset of signal molecules that control tissue specification and differentiation. The transcriptional regulation of cardiomyocyte development has been well established, yet only until recently has it been uncovered that miRNAs participate in the regulatory networks. A subset of miRNAs are either specifically or highly expressed in cardiac muscle, providing an opportunity to understand how gene expression is controlled by miRNAs at the post-transcriptional level in this muscle type. miR-1, miR-133, miR-206, and miR-208 have been found to be muscle-specific, and thus have been called myomiRs. The discovery of myomiRs as a previously unrecognized component in the regulation of gene expression adds an entirely new layer of complexity to our understanding of cardiac muscle development. Investigating myomiRs will not only reveal novel molecular mechanisms of the miRNA-mediated regulatory network in cardiomyocyte development, but also raise new opportunities for therapeutic intervention for cardiovascular disease.