Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs.

Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.

Design and application of circular RNAs with protein-sponge function.

Circular RNAs (circRNAs) are a class of noncoding RNAs, generated from pre-mRNAs by circular splicing of exons and functionally largely uncharacterized. Here we report on the design, expression, and characterization of artificial circRNAs that act as protein sponges, specifically binding and functionally inactivating hnRNP (heterogeneous nuclear ribonucleoprotein) L. HnRNP L regulates alternative splicing, depending on short CA-rich RNA elements. We demonstrate that designer hnRNP L-sponge circRNAs with CA-repeat or CA-rich sequence clusters can efficiently and specifically modulate splicing-regulatory networks in mammalian cells, including alternative splicing patterns and the cellular distribution of a splicing factor. This new strategy can in principle be applied to any RNA-binding protein, opening up new therapeutic strategies in molecular medicine.

Combinatorial recognition of clustered RNA elements by the multidomain RNA-binding protein IMP3.

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

Selective release of circRNAs in platelet-derived extracellular vesicles.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.

Northern Blot Analysis of Circular RNAs.

Northern blotting enables the specific detection and characterization of RNA molecules. Recently, circular RNAs (circRNAs) were described as a new class of cell type-specific noncoding RNAs. With the discovery of many novel circRNAs on the basis of high-throughput sequencing and bioinformatics, a solid biochemical approach is required to directly detect and validate specific circRNA species. Here we give a detailed overview of how different Northern blot methods can be employed to validate specific circRNAs. Different Northern gel and detection systems are introduced, in combination with additional tools for circRNA characterization, such as RNase R and RNase H treatments.

CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.

Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component.

Exon circularization requires canonical splice signals.

Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode.

Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L.

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a multifunctional RNA-binding protein that is involved in many different processes, such as regulation of transcription, translation, and RNA stability. We have previously characterized hnRNP L as a global regulator of alternative splicing, binding to CA-repeat, and CA-rich RNA elements. Interestingly, hnRNP L can both activate and repress splicing of alternative exons, but the precise mechanism of hnRNP L-mediated splicing regulation remained unclear. To analyze activities of hnRNP L on a genome-wide level, we performed individual-nucleotide resolution crosslinking-immunoprecipitation in combination with deep-sequencing (iCLIP-Seq). Sequence analysis of the iCLIP crosslink sites showed significant enrichment of C/A motifs, which perfectly agrees with the in vitro binding consensus obtained earlier by a SELEX approach, indicating that in vivo hnRNP L binding targets are mainly determined by the RNA-binding activity of the protein. Genome-wide mapping of hnRNP L binding revealed that the protein preferably binds to introns and 3' UTR. Additionally, position-dependent splicing regulation by hnRNP L was demonstrated: The protein represses splicing when bound to intronic regions upstream of alternative exons, and in contrast, activates splicing when bound to the downstream intron. These findings shed light on the longstanding question of differential hnRNP L-mediated splicing regulation. Finally, regarding 3' UTR binding, hnRNP L binding preferentially overlaps with predicted microRNA target sites, indicating global competition between hnRNP L and microRNA binding. Translational regulation by hnRNP L was validated for a subset of predicted target 3'UTRs.

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing.

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4-6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4-6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.

Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage.

Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations.

In vitro and in vivo effects on neural crest stem cell differentiation by conditional activation of Runx1 short isoform and its effect on neuropathic pain behavior.

INTRODUCTION: Runx1, a Runt domain transcription factor, controls the differentiation of nociceptors that express the neurotrophin receptor Ret, regulates the expression of many ion channels and receptors, and controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. We investigated whether conditional activation of Runx1 short isoform (Runx1a), which lacks a transcription activation domain, influences differentiation of neural crest stem cells (NCSCs) in vitro and in vivo during development and whether postnatal Runx1a activation affects the sensitivity to neuropathic pain. METHODS: We activated ectopic expression of Runx1a in cultured NCSCs using the Tet-ON gene regulatory system during the formation of neurospheres and analyzed the proportion of neurons and glial cells originating from NCSCs. In in vivo experiments we applied doxycycline (DOX) to pregnant mice (days 8-11), i.e. when NCSCs actively migrate, and examined the phenotype of offsprings. We also examined whether DOX-induced activation of Runx1a in adult mice affects their sensitivity to mechanical stimulation following a constriction injury of the sciatic nerve. RESULTS: Ectopic Runx1a expression in cultured NCSCs resulted in predominantly glial differentiation. Offsprings in which Runx1a had been activated showed retarded growth and displayed megacolon, pigment defects, and dystrophic dorsal root ganglia. In the neuropathic pain model, the threshold for mechanical sensitivity was markedly increased following activation of Runx1a. CONCLUSION: These data suggest that Runx1a has a specific role in NCSC development and that modulation of Runx1a activity may reduce mechanical hypersensitivity associated with neuropathic pain.

Replacement of mouse Sox10 by the Drosophila ortholog Sox100B provides evidence for co-option of SoxE proteins into vertebrate-specific gene-regulatory networks through altered expression.

Neural crest cells and oligodendrocytes as the myelinating glia of the central nervous system exist only in vertebrates. Their development is regulated by complex regulatory networks, of which the SoxE-type high-mobility-group domain transcription factors Sox8, Sox9 and Sox10 are essential components. Here we analyzed by in ovo electroporation in chicken and by gene replacement in the mouse whether the Drosophila ortholog Sox100B can functionally substitute for vertebrate SoxE proteins. Sox100B overexpression in the chicken neural tube led to the induction of neural crest cells as previously observed for vertebrate SoxE proteins. Furthermore, many aspects of neural crest and oligodendrocyte development were surprisingly normal in mice in which the Sox10 coding information was replaced by Sox100B arguing that Sox100B integrates well into the gene-regulatory networks that drive these processes. Our results therefore provide strong evidence for a model in which SoxE proteins were co-opted to these gene-regulatory networks mainly through the acquisition of novel expression patterns. However, later developmental defects in several neural crest derived lineages in mice homozygous for the Sox100B replacement allele indicate that some degree of functional specialization and adaptation of SoxE protein properties have taken place in addition to the co-option event.

HnRNP L-mediated regulation of mammalian alternative splicing by interference with splice site recognition.

Heterogeneous nuclear ribonucleoprotein (hnRNP) L can regulate alternative mRNA splicing in diverse ways, binding to exonic or intronic sites and acting as either an activator or repressor. To investigate the mechanistic basis of hnRNP L-regulated alternative splicing, we focus here on two specific cases of hnRNP L-dependent splice site recognition. First, in the case of TJP1 our microarray data had suggested that exon 20 inclusion is regulated by hnRNP L as a repressor. Here we demonstrate by mutational analysis that exon skipping is mediated by a short silencer sequence consisting of three hnRNP L high-score binding motifs located upstream of the 3' splice site of the regulated exon. UV crosslinking and immunoprecipitation experiments showed that hnRNP L binding interferes with 3' splice site recognition by U2AF65. Second, SLC2A2 contains a CA-repeat sequence close to the 5' splice site of the regulated exon 4. Using psoralen crosslinking, we demonstrate that hnRNP L represses splicing by preventing 5' splice site recognition of the U1 snRNP. In sum, our data provide new insights into the mechanisms of how hnRNP L-bound to intronic sites-regulates exon recognition.

Activation of Krox20 gene expression by Sox10 in myelinating Schwann cells.

The high-mobility group domain transcription factor Sox10 is believed to influence myelination in Schwann cells by directly activating myelin genes and by inducing Krox20 as a pivotal regulator of peripheral myelination. Krox20 induction at this stage is thought to be mediated by the myelinating Schwann cell element 35 kb downstream of the Krox20 transcriptional start site and requires cooperation with Oct6. Here, we prove for the first time in vivo that Schwann cell-specific Krox20 expression indeed depends on Sox10. We also provide evidence that Sox10 functions through multiple, mostly monomeric binding sites in the myelinating Schwann cell element in a manner that should render the enhancer exquisitely sensitive to Sox10 levels. Synergistic activation of the enhancer by Sox10 and Oct6 furthermore does not involve cooperative binding to closely spaced binding sites in defined composite elements. Nevertheless, the POU domain of Oct6 and the high-mobility group domain of Sox10 as the two DNA-binding domains were both essential indicating that each transcription factor has to bind independently to DNA. Whereas the POU domain was the only important region of Oct6, two further Sox10 domains were required for synergistic Krox20 activation. These were the carboxyterminal transactivation domain and the conserved K2 domain in the central portion of Sox10. All required regions are conserved in several closely related POU and Sox proteins thus explaining why Oct6 and Sox10 can be replaced by their relatives during Krox20 induction in myelinating Schwann cells.

Regulation of boundary cap neural crest stem cell differentiation after transplantation.

Success of cell replacement therapies for neurological disorders will depend largely on the optimization of strategies to enhance viability and control the developmental fate of stem cells after transplantation. Once transplanted, stem/progenitor cells display a tendency to maintain an undifferentiated phenotype or differentiate into inappropriate cell types. Gain and loss of function experiments have revealed key transcription factors which drive differentiation of immature stem/progenitor cells toward more mature stages and eventually to full differentiation. An attractive course of action to promote survival and direct the differentiation of transplanted stem cells to a specific cell type would therefore be to force expression of regulatory differentiation molecules in already transplanted stem cells, using inducible gene expression systems which can be controlled from the outside. Here, we explore this hypothesis by employing a tetracycline gene regulating system (Tet-On) to drive the differentiation of boundary cap neural crest stem cells (bNCSCs) toward a sensory neuron fate after transplantation. We induced the expression of the key transcription factor Runx1 in Sox10-expressing bNCSCs. Forced expression of Runx1 strongly increased transplant survival in the enriched neurotrophic environment of the dorsal root ganglion cavity, and was sufficient to guide differentiation of bNCSCs toward a nonpeptidergic nociceptive sensory neuron phenotype both in vitro and in vivo after transplantation. These findings suggest that exogenous activation of transcription factors expression after transplantation in stem/progenitor cell grafts can be a constructive approach to control their survival as well as their differentiation to the desired type of cell and that the Tet-system is a useful tool to achieve this.

Auto- and cross-regulation of the hnRNP L proteins by alternative splicing.

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This "poison exon" is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

SoxE proteins are differentially required in mouse adrenal gland development.

Sry-box (Sox)8, Sox9, and Sox10 are all strongly expressed in the neural crest. Here, we studied the influence of these closely related transcription factors on the developing adrenal medulla as one prominent neural crest derivative. Whereas Sox9 was not expressed, both Sox8 and Sox10 occurred widely in neural crest cells migrating to the adrenal gland and in the gland itself, and they were down-regulated in cells expressing catecholaminergic traits. Sox10-deficient mice lacked an adrenal medulla. The adrenal anlage was never colonized by neural crest cells, which failed to specify properly at the dorsal aorta and died apoptotically during migration. Furthermore, mutant neural crest cells did not express Sox8. Strong adrenal phenotypes were also observed when the Sox10 dimerization domain was inactivated or when a transactivation domain in the central portion was deleted. Sox8 in contrast had only minimal influence on adrenal gland development. Phenotypic consequences became only visible in Sox8-deficient mice upon additional deletion of one Sox10 allele. Replacement of Sox10 by Sox8, however, led to significant rescue of the adrenal medulla, indicating that functional differences between the two related Sox proteins contribute less to the different adrenal phenotypes of the null mutants than dependence of Sox8 expression on Sox10.

Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis.

Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/A-rich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.

Hypomorphic Sox10 alleles reveal novel protein functions and unravel developmental differences in glial lineages.

The transcription factor Sox10 regulates early neural crest development, specification of neural crest-derived lineages and terminal differentiation of oligodendrocytes in the central nervous system. Here, we generated two novel hypomorphic Sox10 alleles in the mouse. Mutant mice either expressed a Sox10 protein with a triple alanine substitution in the dimerization domain, or a Sox10 protein with a deletion in the central portion that we define as a cell-specific transactivation domain. Phenotypic analysis revealed important roles for a functional dimerization domain and the newly defined novel transactivation domain in melanocyte and enteric nervous system development, whereas early neural crest development and oligodendrocyte differentiation were surprisingly little disturbed in both mutants. Unique requirements were additionally detected for the novel transactivation domain in satellite glia differentiation and during Schwann cell myelination, whereas DNA-dependent dimerization was needed for immature Schwann cells to enter the promyelinating stage. These two hypomorphic alleles thus uncover novel functions of Sox10 in satellite glia and Schwann cells during late developmental stages and reveal important developmental differences between these two types of peripheral glia and oligodendrocytes regarding their reliance on Sox10.

Replacement of the Sox10 transcription factor by Sox8 reveals incomplete functional equivalence.

Sox8 and Sox10 are two closely related transcription factors of the Sox protein family with overlapping expression patterns during development. They are believed to perform very similar functions because several developmental processes, including enteric nervous system development and oligodendrocyte differentiation, are regulated by both Sox proteins. To analyze the extent of functional equivalence between the two Sox proteins, we employed targeted mutagenesis to replace Sox10 with Sox8 in the mouse. In mice that expressed Sox8 instead of Sox10, Sox10 deficiency was phenotypically rescued to different extents in affected tissues. Whereas development of glial cells and neurons in the sensory and sympathetic parts of the peripheral nervous system was almost normal when Sox10 was replaced by Sox8, melanocyte development was as defective as in Sox10-deficient mice. The ability of Sox8 to rescue the defects in enteric nervous system development and oligodendrocyte differentiation of Sox10-deficient mice was limited. We conclude that the extent of functional equivalence depends on the tissue and that, despite their relatedness, Sox8 and Sox10 have more unique functions than previously appreciated.