FUS-dependent microRNA deregulations identify TRIB2 as a druggable target for ALS motor neurons.

MicroRNAs (miRNAs) modulate mRNA expression, and their deregulation contributes to various diseases including amyotrophic lateral sclerosis (ALS). As fused in sarcoma (FUS) is a causal gene for ALS and regulates biogenesis of miRNAs, we systematically analyzed the miRNA repertoires in spinal cords and hippocampi from ALS-FUS mice to understand how FUS-dependent miRNA deregulation contributes to ALS. miRNA profiling identified differentially expressed miRNAs between different central nervous system (CNS) regions as well as disease states. Among the up-regulated miRNAs, miR-1197 targets the pro-survival pseudokinase Trib2. A reduced TRIB2 expression was observed in iPSC-derived motor neurons from ALS patients. Pharmacological stabilization of TRIB2 protein with a clinically approved cancer drug rescues the survival of iPSC-derived human motor neurons, including those from a sporadic ALS patient. Collectively, our data indicate that miRNA profiling can be used to probe the molecular mechanisms underlying selective vulnerability, and TRIB2 is a potential therapeutic target for ALS.

A novel eukaryotic RdRP-dependent small RNA pathway represses antiviral immunity by controlling an ERK pathway component in the black-legged tick.

Small regulatory RNAs (sRNAs) are involved in antiviral defense and gene regulation. Although roles of RNA-dependent RNA Polymerases (RdRPs) in sRNA biology are extensively studied in nematodes, plants and fungi, understanding of RdRP homologs in other animals is still lacking. Here, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We find abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP1-dependent sRNAs possess 5'-monophosphates and are mainly derived from RNA polymerase III-transcribed genes and repetitive elements. Knockdown of some RdRP homologs misregulates genes including RNAi-related genes and the regulator of immune response Dsor1. Sensor assays demonstrate that Dsor1 is downregulated by RdRP1 through the 3'UTR that contains a target site of RdRP1-dependent repeat-derived sRNAs. Consistent with viral gene repression by the RNAi mechanism using virus-derived small interfering RNAs, viral transcripts are upregulated by AGO knockdown. On the other hand, RdRP1 knockdown unexpectedly results in downregulation of viral transcripts. This effect is dependent on Dsor1, suggesting that antiviral immunity is enhanced by RdRP1 knockdown through Dsor1 upregulation. We propose that tick sRNA pathways control multiple aspects of immune response via RNAi and regulation of signaling pathways.

Upregulated Blood miR-150-5p in Alzheimer's Disease Dementia Is Associated with Cognition, Cerebrospinal Fluid Amyloid-ß, and Cerebral Atrophy.

BACKGROUND: There is an urgent need for noninvasive, cost-effective biomarkers for Alzheimer's disease (AD), such as blood-based biomarkers. They will not only support the clinical diagnosis of dementia but also allow for timely pharmacological and nonpharmacological interventions and evaluations. OBJECTIVE: To identify and validate a novel blood-based microRNA biomarker for dementia of the Alzheimer's type (DAT). METHODS: We conducted microRNA sequencing using peripheral blood mononuclear cells isolated from a discovery cohort and validated the identified miRNAs in an independent cohort and AD postmortem tissues. miRNA correlations with AD pathology and AD clinical-radiological imaging were conducted. We also performed bioinformatics and cell-based assay to identify miRNA target genes. RESULTS: We found that miR-150-5p expression was significantly upregulated in DAT compared to mild cognitive impairment and healthy subjects. Upregulation of miR-150-5p was observed in AD hippocampus. We further found that higher miR-150-5p levels were correlated with the clinical measures of DAT, including lower global cognitive scores, lower CSF Aß42, and higher CSF total tau. Interestingly, we observed that higher miR-150-5p levels were associated with MRI brain volumes within the default mode and executive control networks, two key networks implicated in AD. Furthermore, pathway analysis identified the targets of miR-150-5p to be enriched in the Wnt signaling pathway, including programmed cell death 4 (PDCD4). We found that PDCD4 was downregulated in DAT blood and was downregulated by miR-150-5p at both the transcriptional and protein levelsConclusion:Our findings demonstrated that miR-150-5p is a promising clinical blood-based biomarker for DAT.

Incorporating 2-Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A-U Pairs and for Targeting a MicroRNA Hairpin Precursor.

Chemically modified short peptide nucleic acids (PNAs) recognize RNA duplexes under near physiological conditions by major-groove PNA·RNA-RNA triplex formation and show great promise for the development of RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T·A-U and U·A-U base triple formation. Incorporation of a modified nucleobase, 2-thiouracil (s2U), into triplex-forming oligonucleotides stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease in the dehydration energy penalty for triplex formation as well as a decrease in the pKa of the N3 atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s2U into short PNAs, followed by binding studies of a series of s2U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis and thermal melting experiments that s2U and L incorporated into dsRNA-binding PNAs (dbPNAs) enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to the DNA duplex. Combining s2U and L modifications in dbPNAs facilitates enhanced recognition of dsRNAs and maintains selective binding to dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s2U- and L-modified dbPNAs (8-mer, with a molecular mass of ∼2.3 kDa) in the inhibition of the pre-microRNA-198 maturation in a substrate-specific manner. Thus, s2U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions.

General Recognition of U-G, U-A, and C-G Pairs by Double-Stranded RNA-Binding PNAs Incorporated with an Artificial Nucleobase.

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA-RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson-Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.

Hidden sequence specificity in loading of single-stranded RNAs onto Drosophila Argonautes.

Argonaute proteins play important roles in gene regulation with small RNAs (sRNAs) serving as guides to targets. Argonautes are believed to bind sRNAs in a sequence non-specific manner. However, we recently discovered that Argonautes selectively load endogenous single-stranded (ss) RNAs, suggesting that Argonaute loading may conform to sequence specificity. To identify sequences preferred for Argonaute loading, we have developed HIgh-throughput Sequencing mediated Specificity Analysis (HISSA). HISSA allows massively parallel analysis of RNA binding efficiency by using randomized oligos in in vitro binding assays and quantifying RNAs by deep-sequencing. We chose Drosophila as a model system to take advantage of the presence of two biochemically distinct Argonautes, AGO1 and AGO2. Our results revealed AGO2 loading to be strongly favored by G-rich sequences. In contrast, AGO1 showed an enrichment of the 'GAC' motif in loaded species. Reanalysis of published sRNA sequencing data from fly tissues detected enrichment of the GAC motif in ssRNA-derived small RNAs in the immunopurified AGO1-complex under certain conditions, suggesting that the sequence preference of AGO1-loading may influence the repertoire of AGO1-bound endogenous sRNAs. Finally, we showed that human Ago2 also exhibited selectivity in loading ssRNAs in cell lysates. These findings may have implications for therapeutic ssRNA-mediated gene silencing.

Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development.

Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, we profile a time course of mature and pri-miRNAs in Drosophila embryos and reveal the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. We found that 5' nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, we show that rapid degradation of miR-3/-309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.

Heterochromatin protein 1a functions for piRNA biogenesis predominantly from pericentric and telomeric regions in Drosophila.

In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila. Here we show that HP1a germline knockdown (HP1a-GLKD) leads to a reduction in the total and Piwi-bound piRNAs mapping to clusters and transposons insertions, predominantly in the regions close to telomeres and centromeres, resulting in derepression of a limited number of transposons from these regions. In addition, HP1a-GLKD increases the splicing of transcripts arising from clusters in above regions, suggesting HP1a also functions upstream to piRNA processing. Evolutionarily old transposons enriched in the pericentric regions exhibit significant loss in piRNAs targeting these transposons upon HP1a-GLKD. Our study suggests that HP1a functions to repress transposons in a chromosomal compartmentalised manner.

Gateway to Understanding Argonaute Loading of Single-Stranded RNAs: Preparation of Deep Sequencing Libraries with In Vitro Loading Samples.

Identification of sequences preferred by individual RNA-binding proteins (RBPs) has been accelerated by recent advances in the quantitative analysis of protein-RNA interactions on a massive scale, and such experiments have even revealed hidden sequence specificity of RBPs that were assumed to be non-specific. Argonaute (AGO) proteins bind diverse guide small RNAs and were believed to have no sequence specificity besides the preference for particular bases at the 5' nucleotide. However, we recently showed that short single-stranded RNAs (ssRNAs) are loaded to AGOs in vivo and in cell extracts with detectable sequence preferences. To study the sequence specificity, we established a protocol for preparing the oligo-specific deep-sequencing library. The protocol includes in vitro loading assay that uses RNA oligos containing randomized nucleotides at the first five positions and also splinted-ligation that specifically amplifies the introduced oligo RNA species from a complex mixture of endogenous small RNAs and exogenously introduced RNA oligos. With the current sequencing depth, this procedure will allow quantitative profiling of interactions between the AGO and ~1000 ssRNA species with different sequences. The method would aid in studying the mechanism behind the selective loading of ssRNAs to AGOs and may potentially be applied to study interactions between RNA and other RNA-binding proteins.

Switches in Dicer Activity During Oogenesis and Early Development.

Dicer is a versatile protein regulating diverse biological processes via the production of multiple classes of small regulatory RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs). In this chapter, we will discuss roles for Dicer in driving temporal changes in activity of individual small RNA classes to support oogenesis and early embryogenesis. Genetic strategies that perturb particular functions of Dicer family proteins, such as ablation of individual Dicer paralogs or their binding partners as well as introduction of point mutations to individual domains, allowed the dissection of Dicer functions in diverse small RNA pathways. Evolutionary conservation and divergence of the mechanisms highlight the importance of Dicer versatility in supporting rapid changes in gene expression during oogenesis and early development. Furthermore, we will discuss potential roles of Dicer in transgenerational inheritance of small RNA-mediated gene regulation.

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site.

In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only ∼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.

Regulatory RNAs discovered in unexpected places.

Recent studies have discovered both small and long noncoding RNAs (ncRNAs) encoded in unexpected places. These ncRNA genes were surprises at the time of their discovery, but many quickly became well-accepted families of functional regulatory RNA species. Even after years of extensive gene annotation studies using high-throughput sequencing technologies, new types of ncRNA genes continue to be discovered in unexpected places. We highlight ncRNAs that have atypical structures and that are encoded in what are generally considered 'junk' sequences, such as spacers and introns. We also discuss current bottlenecks in the approaches for identifying novel ncRNAs and the possibility that many remain to be discovered.

Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor.

Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genome-wide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibits sequence preference for 3'-terminal AG, a defining mirtron characteristic. Our work supports the notion that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, we detect preferential activity of Tailor on 3'-G canonical pre-miRNAs, and specific depletion of such loci from the pool of conserved miRNAs. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs.

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA.

Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the pre-rRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5' and 3' ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha-Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼ 100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms.

Adaptive regulation of testis gene expression and control of male fertility by the Drosophila hairpin RNA pathway. [Corrected].

Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-ß in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.

A signaling-induced switch in dicer localization and function.

In this issue of Developmental Cell, Drake and colleagues (2014) report that Ras signaling results in Dicer phosphorylation, which induces its nuclear localization and modulates its function. This regulatory strategy, conserved in mammals, allows dynamic control of microRNA function required for Caenorhabditis elegans germline development and oogenesis.

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Argonaute-dependent small RNAs derived from single-stranded, non-structured precursors.

A general feature of Argonaute-dependent small RNAs is their base-paired precursor structures, and precursor duplex structures are often required for confident annotation of miRNA genes. However, this rule has been broken by discoveries of functional small RNA species whose precursors lack a predictable double-stranded (ds-) RNA structure, arguing that duplex structures are not prerequisite for small RNA loading to Argonautes. The biological significance of single-stranded (ss-) RNA loading has been recognized particularly in systems where active small RNA amplification mechanisms are involved, because even a small amount of RNA molecules can trigger the production of abundant RNA species leading to profound biological effects. However, even in the absence of small RNA amplification mechanisms, recent studies have demonstrated that potent gene silencing can be achieved using chemically modified synthetic ssRNAs that are resistant to RNases in mice. Therefore, such ssRNA-mediated gene regulation may have broader roles than previously recognized, and the findings have opened the door for further research to optimize the design of ss-siRNAs toward future pharmaceutical and biomedical applications of gene silencing technologies. In this review, we will summarize studies about endogenous ssRNA species that are bound by Argonaute proteins and how ssRNA precursors are recognized by various small RNA pathways.

Argonaute reformatting.

In this issue of Molecular Cell, De et al. (2013) report that highly complementary targets promote release of small RNAs from effector Argonaute complexes, thus providing mechanistic insights into regulation of small RNA stability and implications for siRNA design.

Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals.

In the canonical animal microRNA (miRNA) pathway, Drosha generates ∼60- to 70-nucleotide pre-miRNA hairpins that are cleaved by Dicer into small RNA duplexes that load into Argonaute proteins, which retain a single mature strand in the active complex. The terminal loops of some miRNA hairpins regulate processing efficiency, but once liberated by Dicer, they are generally considered nonfunctional by-products. Here, we show that specific miRNA loops accumulate in effector Argonaute complexes in Drosophila and mediate miRNA-type repression. This was unexpected, since endogenous loading of Argonaute proteins was believed to occur exclusively via small RNA duplexes. Using in vitro assays, which recapitulate Argonaute-specific loop loading from synthetic pre-miRNAs and even single-stranded oligoribonucleotides corresponding to miRNA loops, we reveal that the loop-loading mechanism is distinct from duplex loading. We also show that miRNA loops loaded into the miRNA effector AGO1 are subject to 3' resection, and structure-function analyses indicate selectivity of loop loading. Finally, we demonstrate that select miRNA loops in mammals are similarly loaded into Argonaute complexes and direct target repression. Altogether, we reveal a conserved mechanism that yields functional RNAs from miRNA loop regions, broadening the repertoire of Argonaute-dependent regulatory RNAs and providing evidence for functionality of endogenous ssRNA species.