SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Intragenic MicroRNAs Autoregulate Their Host Genes in Both Direct and Indirect Ways-A Cross-Species Analysis.

MicroRNAs (miRNAs) function as master switches for post-transcriptional gene expression. Their genes are either located in the extragenic space or within host genes, but these intragenic miRNA::host gene interactions are largely enigmatic. The aim of this study was to investigate the location and co-regulation of all to date available miRNA sequences and their host genes in an unbiased computational approach. The majority of miRNAs were located within intronic regions of protein-coding and non-coding genes. These intragenic miRNAs exhibited both increased target probability as well as higher target prediction scores as compared to a model of randomly permutated genes. This was associated with a higher number of miRNA recognition elements for the hosted miRNAs within their host genes. In addition, strong indirect autoregulation of host genes through modulation of functionally connected gene clusters by intragenic miRNAs was demonstrated. In addition to direct miRNA-to-host gene targeting, intragenic miRNAs also appeared to interact with functionally related genes, thus affecting their host gene function through an indirect autoregulatory mechanism. This strongly argues for the biological relevance of autoregulation not only for the host genes themselves but, more importantly, for the entire gene cluster interacting with the host gene.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease.

Chronic kidney disease (CKD) is a progressive pathological condition marked by a gradual loss of kidney function. Treatment of CKD is most effective when diagnosed at an early stage and patients are still asymptomatic. However, current diagnostic biomarkers (e.g., serum creatinine and urine albumin) are insufficient for prediction of the pathogenesis of the disease. To address this need, we applied a cell-SELEX (systematic evolution of ligands by exponential enrichment) approach and identified a series of DNA aptamers, which exhibit high affinity and selectivity for cytokine-stimulated cells, resembling some aspects of a CKD phenotype. The cell-SELEX approach was driven toward the enrichment of aptamers that internalize via the endosomal pathway by isolating the endosomal fractions in each selection cycle. Indeed, we demonstrated co-localization of selected aptamers with lysosomal-associated membrane protein 1 (LAMP-1), a late endosomal and lysosomal marker protein, by fluorescence in situ hybridization. These findings are consistent with binding and subsequent internalization of the aptamers into cytokine-stimulated cells. Thus, our study sets the stage for applying selected DNA aptamers as theragnostic reagents for the development of targeted therapies to combat CKD.

Circulating miRNAs as biomarkers of kidney disease.

Interest in microRNAs (miRNAs) has dramatically increased in recent years not only because they regulate mRNA expression, and thus many physiological or pathophysiological processes, but also because they could serve as biomarkers. Next to analysis of tissue miRNA expression, measurement in body fluids such as blood or urine is attractive because miRNA in microvesicles or bound to protein is very stable. Currently it is unclear whether these circulating miRNAs are tissue and disease specific or represent more general pathologies like inflammation. In addition pre-analytical sample handling and variable analysis techniques affect the results and thus much more work needs to be done before one can draw a final conclusion about their clinical utility.

A novel but frequent variant in LPA KIV-2 is associated with a pronounced Lp(a) and cardiovascular risk reduction.

AIMS: Lp(a) concentrations represent a major cardiovascular risk factor and are almost entirely controlled by one single locus (LPA). However, many genetic factors in LPA governing the enormous variance of Lp(a) levels are still unknown. Since up to 70% of the LPA coding sequence are located in a difficult to access hypervariable copy number variation named KIV-2, we hypothesized that it may contain novel functional variants with pronounced effects on Lp(a) concentrations. We performed a large scale mutation analysis in the KIV-2 using an extreme phenotype approach. METHODS AND RESULTS: We compiled an discovery set of 123 samples showing discordance between LPA isoform phenotype and Lp(a) concentrations and controls. Using ultra-deep sequencing, we identified a splice site variant (G4925A) in preferential association with the smaller LPA isoforms. Follow-up in a European general population (n = 2892) revealed an exceptionally high carrier frequency of 22.1% in the general population. The variant explains 20.6% of the Lp(a) variance in carriers of low molecular weight (LMW) apo(a) isoforms (P = 5.75e-38) and reduces Lp(a) concentrations by 31.3 mg/dL. Accordingly the odds ratio for cardiovascular disease was reduced from 1.39 [95% confidence interval (CI): 1.17-1.66, P = 1.89e-04] for wildtype LMW individuals to 1.19 [95%CI: 0.92; 1.56, P = 0.19] in LMW individuals who were additionally positive for G4925A. Functional studies point towards a reduction of splicing efficiency by this novel variant. CONCLUSION: A highly frequent but until now undetected variant in the LPA KIV-2 region is strongly associated with reduced Lp(a) concentrations and reduced cardiovascular risk in LMW individuals.

MicroRNA-Mediated Rescue of Fear Extinction Memory by miR-144-3p in Extinction-Impaired Mice.

BACKGROUND: MicroRNA (miRNA)-mediated control of gene expression suggests that miRNAs are interesting targets and/or biomarkers in the treatment of anxiety- and trauma-related disorders, where often memory-associated gene expression is adversely affected. METHODS: The role of miRNAs in the rescue of impaired fear extinction was assessed using the 129S1/SvlmJ (S1) mouse model of impaired fear extinction. miRNA microarray analysis, reverse transcription polymerase chain reaction, fluorescent in situ hybridization, lentiviral overexpression, and Luciferase reporter assays were used to gain insight into the mechanisms underlying miRNA-mediated normalization of deficient fear extinction. RESULTS: Rescuing impaired fear extinction via dietary zinc restriction was associated with differential expression of miRNAs in the amygdala. One candidate, miR-144-3p, robustly expressed in the basolateral amygdala, showed specific extinction-induced, but not fear-induced, increased expression in both extinction-rescued S1 mice and extinction-intact C57BL/6 (BL6) mice. miR-144-3p upregulation and effects on subsequent behavioral adaption was assessed in S1 and BL6 mice. miR-144-3p overexpression in the basolateral amygdala rescued impaired fear extinction in S1 mice, led to enhanced fear extinction acquisition in BL6 mice, and furthermore protected against fear renewal in BL6 mice. miR-144-3p targets a number of genes implicated in the control of plasticity-associated signaling cascades, including Pten, Spred1, and Notch1. In functional interaction studies, we revealed that the miR-144-3p target, PTEN, colocalized with miR-144-3p in the basolateral amygdala and showed functional downregulation following successful fear extinction in S1 mice. CONCLUSIONS: These findings identify a fundamental role of miR-144-3p in the rescue of impaired fear extinction and suggest this miRNA as a viable target in developing novel treatments for posttraumatic stress disorder and related disorders.

Identification of urinary exosomal noncoding RNAs as novel biomarkers in chronic kidney disease.

In chronic kidney disease (CKD), the decline in the glomerular filtration rate is associated with increased morbidity and mortality and thus poses a major challenge for healthcare systems. While the contribution of tissue-derived miRNAs and mRNAs to CKD progression has been extensively studied, little is known about the role of urinary exosomes and their association with CKD. Exosomes are small, membrane-derived endocytic vesicles that contribute to cell-to-cell communication and are present in various body fluids, such as blood or urine. Next-generation sequencing approaches have revealed that exosomes are enriched in noncoding RNAs and thus exhibit great potential for sensitive nucleic acid biomarkers in various human diseases. Therefore, in this study we aimed to identify urinary exosomal ncRNAs as novel biomarkers for diagnosis of CKD. Since up to now most approaches have focused on the class of miRNAs, we extended our analysis to several other noncoding RNA classes, such as tRNAs, tRNA fragments (tRFs), mitochondrial tRNAs, or lincRNAs. For their computational identification from RNA-seq data, we developed a novel computational pipeline, designated as ncRNASeqScan. By these analyses, in CKD patients we identified 30 differentially expressed ncRNAs, derived from urinary exosomes, as suitable biomarkers for early diagnosis. Thereby, miRNA-181a appeared as the most robust and stable potential biomarker, being significantly decreased by about 200-fold in exosomes of CKD patients compared to healthy controls. Using a cell culture system for CKD indicated that urinary exosomes might indeed originate from renal proximal tubular epithelial cells.

mRNA modifications: Dynamic regulators of gene expression?

The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), pseudouridine (Ψ) and N(1)-methyladenosine (m(1)A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression.

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications.

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m(5)C, m(6)A, Ψ or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs.

Cell-SELEX: in vitro selection of synthetic small specific ligands.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is an in vitro process enabling selection of nucleic acid molecules binding to target ligands with high binding affinity and specificity. The selection process involves several rounds of two successive steps: (1) binding of the oligonucleotides to the target under stringent conditions and (2) amplification of the target-bound nucleic acids by polymerase chain reaction. Using this strategy, RNA or DNA aptamers are selected upon recognition and binding to specific surface structures of the target. Aptamers generated during the final rounds of selection can be notably used in applications dedicated to diagnosis of diseases or therapeutic approaches.

Generation of a neuro-specific microarray reveals novel differentially expressed noncoding RNAs in mouse models for neurodegenerative diseases.

We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson's disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer's disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer's disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs.

microRNAs in nociceptive circuits as predictors of future clinical applications.

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals.

Expression Profiling of a Heterogeneous Population of ncRNAs Employing a Mixed DNA/LNA Microarray.

Mammalian transcriptomes mainly consist of non protein coding RNAs. These ncRNAs play various roles in all cells and are involved in multiple regulation pathways. More recently, ncRNAs have also been described as valuable diagnostic tools. While RNA-seq approaches progressively replace microarray-based technologies for high-throughput expression profiling, they are still not routinely used in diagnostic. Microarrays, on the other hand, are more widely used for diagnostic profiling, especially for very small ncRNA (e.g., miRNAs), employing locked nucleic acid (LNA) arrays. However, LNA microarrays are quite expensive for high-throughput studies targeting longer ncRNAs, while DNA arrays do not provide satisfying results for the analysis of small RNAs. Here, we describe a mixed DNA/LNA microarray platform, where directly labeled small and longer ncRNAs are hybridized on LNA probes or custom DNA probes, respectively, enabling sensitive and specific analysis of a complex RNA population on a unique array in one single experiment. The DNA/LNA system, requiring relatively low amounts of total RNA, which complies with diagnostic references, was successfully applied to the analysis of differential ncRNA expression in mouse embryonic stem cells and adult brain cells.

Identification of differentially expressed non-coding RNAs in embryonic stem cell neural differentiation.

Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18-30 nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.