Megalencephalic leukoencephalopathy with subcortical cysts: a variant update and review of the literature.

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.

Xrn1-resistant RNA motifs are disseminated throughout the RNA virome and are able to block scanning ribosomes.

RNAs that are able to prevent degradation by the 5'-3' exoribonuclease Xrn1 have emerged as crucial structures during infection by an increasing number of RNA viruses. Several plant viruses employ the so-called coremin motif, an Xrn1-resistant RNA that is usually located in 3' untranslated regions. Investigation of its structural and sequence requirements has led to its identification in plant virus families beyond those in which the coremin motif was initially discovered. In this study, we identified coremin-like motifs that deviate from the original in the number of nucleotides present in the loop region of the 5' proximal hairpin. They are present in a number of viral families that previously did not have an Xrn1-resistant RNA identified yet, including the double-stranded RNA virus families Hypoviridae and Chrysoviridae. Through systematic mutational analysis, we demonstrated that a coremin motif carrying a 6-nucleotide loop in the 5' proximal hairpin generally requires a YGNNAD consensus for stalling Xrn1, similar to the previously determined YGAD consensus required for Xrn1 resistance of the original coremin motif. Furthermore, we determined the minimal requirements for the 3' proximal hairpin. Since some putative coremin motifs were found in intergenic regions or coding sequences, we demonstrated their capacity for inhibiting translation through an in vitro ribosomal scanning inhibition assay. Consequently, this study provides a further expansion on the number of viral families with known Xrn1-resistant elements, while adding a novel, potentially regulatory function for this structure.

Investigating the correlation between Xrn1-resistant RNAs and frameshifter pseudoknots.

Xrn1-resistant RNA structures are multifunctional elements employed by an increasing number of RNA viruses. One of such elements is the coremin motif, discovered in plant virus RNAs, of which the structure has been hypothesized to form a yet unelucidated pseudoknot. Recently, the coremin motif was shown to be capable of stalling not only Xrn1, but scanning ribosomes as well. Following that observation, in this study we demonstrate that the coremin motif can promote -1 ribosomal frameshifting, similar to better-characterized viral frameshifting pseudoknots. Since this function was lost in concert with substitutions that were known to disturb Xrn1-resistance, we developed a frameshifting screen for finding novel Xrn1-resistant RNAs by randomizing parts of the coremin motif. This yielded new insights into the coremin motif structure, as Xrn1-resistant variations were identified that more clearly indicate a pseudoknot interaction. In addition, we show that the Xrn1-resistant RNA of Zika virus promotes frameshifting as well, while known -1 programmed ribosomal frameshifting pseudoknots do not stall Xrn1, suggesting that promoting frameshifting is a universal characteristic of Xrn1-resistant RNAs, but that Xrn1-resistance requires more than just a frameshifting pseudoknot.

Role of an RNA pseudoknot involving the polyA tail in replication of Pepino mosaic potexvirus and related plant viruses.

Pepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome with a 5'-methylguanosine cap and a 3'-polyA tail. Previously, using partially-purified viral RNA polymerase important secondary structures within the 3'-untranslated region (UTR) of PepMV RNA were identified. Here we show that an RNA pseudoknot can be formed in the 3'-UTR that includes part of the polyA tail. Using protoplasts, we demonstrate that the pseudoknot is required for replication of PepMV RNA. Mutational analysis and native gel electrophoresis further show that the pseudoknot is stabilized by UAU base triples, as is the human telomerase RNA pseudoknot. The presence of a pseudoknot in several other members of the Alpha- and Betaflexiviridae is supported by covariance analysis and native gel electrophoresis of other potexvirus, capillovirus and trichovirus RNAs. The ubiquitous presence of the pseudoknot in viruses of the Betaflexiviridae, suggests that the pseudoknot is a typical trait of the Betaflexiviridae that may have been adopted by many potexviruses during evolution.

Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail.

Alphaviruses, such as the Sindbis virus and the Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates, including humans. A conserved sequence element (CSE) of ∼19 nt in the 3' noncoding region is important for replication. Despite extensive mutational analysis of the CSE, no comprehensive model of this element exists to date. Here, it is shown that the CSE can form an RNA pseudoknot with part of the poly(A) tail and is similar to the human telomerase pseudoknot with which it shares 17 nt. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of the Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE.

Structural phylogenetic analysis reveals lineage-specific RNA repetitive structural motifs in all coronaviruses and associated variations in SARS-CoV-2.

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA-protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5'-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5'-gUUYCGUc-3' RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5'-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5'-agc-3'/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5'-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5'-gUUUCGUc-3' motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity.

Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts.

A database of flavivirus RNA structures with a search algorithm for pseudoknots and triple base interactions.

MOTIVATION: The Flavivirus genus includes several important pathogens, such as Zika, dengue and yellow fever virus. Flavivirus RNA genomes contain a number of functionally important structures in their 3' untranslated regions (3'UTRs). Due to the diversity of sequences and topologies of these structures, their identification is often difficult. In contrast, predictions of such structures are important for understanding of flavivirus replication cycles and development of antiviral strategies. RESULTS: We have developed an algorithm for structured pattern search in RNA sequences, including secondary structures, pseudoknots and triple base interactions. Using the data on known conserved flavivirus 3'UTR structures, we constructed structural descriptors which covered the diversity of patterns in these motifs. The descriptors and the search algorithm were used for the construction of a database of flavivirus 3'UTR structures. Validating this approach, we identified a number of domains matching a general pattern of exoribonuclease Xrn1-resistant RNAs in the growing group of insect-specific flaviviruses. AVAILABILITY AND IMPLEMENTATION: The Leiden Flavivirus RNA Structure Database is available at https://rna.liacs.nl. The search algorithm is available at https://github.com/LeidenRNA/SRHS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Conserved structural RNA domains in regions coding for cleavage site motifs in hemagglutinin genes of influenza viruses.

The acquisition of a multibasic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein is the main determinant of the conversion of low pathogenic avian influenza viruses into highly pathogenic strains, facilitating HA cleavage and virus replication in a broader range of host cells. In nature, substitutions or insertions in HA RNA genomic segments that code for multiple basic amino acids have been observed only in the HA genes of two out of sixteen subtypes circulating in birds, H5 and H7. Given the compatibility of MBCS motifs with HA proteins of numerous subtypes, this selectivity was hypothesized to be determined by the existence of specific motifs in HA RNA, in particular structured domains. In H5 and H7 HA RNAs, predictions of such domains have yielded alternative conserved stem-loop structures with the cleavage site codons in the hairpin loops. Here, potential RNA secondary structures were analyzed in the cleavage site regions of HA segments of influenza viruses of different types and subtypes. H5- and H7-like stem-loop structures were found in all known influenza A virus subtypes and in influenza B and C viruses with homology modeling. Nucleotide covariations supported this conservation to be determined by RNA structural constraints that are stronger in the domain-closing bottom stems as compared to apical parts. The structured character of this region in (sub-)types other than H5 and H7 indicates its functional importance beyond the ability to evolve toward an MBCS responsible for a highly pathogenic phenotype.

Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system.

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.

Performing DNA nanotechnology operations on a zebrafish.

Nanoscale engineering of surfaces is becoming an indispensable technique to modify membranes and, thus cellular behaviour. Here, such membrane engineering related was explored on the surface of a living animal using DNA nanotechnology. We demonstrate the immobilization of oligonucleotides functionalized with a membrane anchor on 2 day old zebrafish. The protruding single-stranded DNA on the skin of zebrafish served as a handle for complementary DNAs, which allowed the attachment of small molecule cargo, liposomes and dynamic relabeling by DNA hybridization protocols. Robust anchoring of the oligonucleotides was proven as DNA-based amplification processes were successfully performed on the outer membrane of the zebrafish enabling the multiplication of surface functionalities from a single DNA-anchoring unit and the dramatic improvement of fluorescent labeling of these animals. As zebrafish are becoming an alternative to animal models in drug development, toxicology and nanoparticles characterization, we believe the platform presented here allows amalgamation of DNA nanotechnology tools with live animals and this opens up yet unexplored avenues like efficient bio-barcoding as well as in vivo tracking.

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles.

Protein delivery into the cytosol of cells is a challenging topic in the field of nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipopeptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation.

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.

Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes.

The influenza A virus genome consists of eight RNA segments. RNA structures within these segments and complementary (cRNA) and protein-coding mRNAs may play a role in virus replication. Here, conserved putative secondary structures that impose significant evolutionary constraints on the gene segment encoding the surface glycoprotein hemagglutinin (HA) were investigated using available sequence data on tens of thousands of virus strains. Structural constraints were identified by analysis of covariations of nucleotides suggested to be paired by structure prediction algorithms. The significance of covariations was estimated by mutual information calculations and tracing multiple covariation events during virus evolution. Covariation patterns demonstrated that structured domains in HA RNAs were mostly subtype-specific, whereas some structures were conserved in several subtypes. The influence of RNA folding on virus replication was studied by plaque assays of mutant viruses with disrupted structures. The results suggest that over the whole length of the HA segment there are local structured domains which contribute to the virus fitness but individually are not essential for the virus. Existence of subtype-specific structured regions in the segments of the influenza A virus genome is apparently an important factor in virus evolution and reassortment of its genes.

Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes.

Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and invivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivo applications.

Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model.

The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery.

Monitoring ribosomal frameshifting as a platform to screen anti-riboswitch drug candidates.

Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target.

RNA structural constraints in the evolution of the influenza A virus genome NP segment.

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

G-quadruplexes within prion mRNA: the missing link in prion disease?

Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrP(C) to infectious PrP(Sc) or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrP(C) have been isolated and found to form G-quadruplexes (G4s). PrP(C) binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrP(Sc). Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed.