Use of steric blocking antisense oligonucleotides for the targeted inhibition of junction containing precursor microRNAs.

Antisense oligonucleotides (ASOs) are widely used as therapeutics for neurodegenerative diseases, cancers, and virus infections. One class of ASOs functions to enhance protein expression by sequestering the mature microRNA (miRNA) in a double-stranded structure within the RNA-induced silencing complex (RISC). An alternative approach for the targeted control of gene expression is to use ASOs that bind to the pre-elements of miRNAs (pre-miRNAs) and modulate their enzymatic processing. Here, we demonstrate that ASOs can be used to disrupt a specific structural feature, "junction," within pre-miR-31 that is important in directing efficient processing by the Dicer/TRBP complex. Furthermore, we extend and validate this strategy to pre-miR-144, which has a similar junction-dependent structure-function relationship. We found that a significant number of human pre-miRNAs are predicted to contain junctions, and validated our ASO approach on several members of this group. Importantly, we also verified the application of junction-targeting ASOs for the specific inhibition of pre-miRNA processing in cell. Our study reemphasizes the important roles of RNA structure in regulating Dicer/TRBP processing of pre-miRNAs and provides the framework to develop structure-informed ASOs that serve to inhibit miRNA production.

Structure of pre-miR-31 reveals an active role in Dicer-TRBP complex processing.

As an essential posttranscriptional regulator of gene expression, microRNA (miRNA) levels must be strictly maintained. The biogenesis of many miRNAs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers, and interestingly, its biogenesis is not known to be regulated by protein-binding partners. Therefore, the intrinsic structural properties of the precursor element of miR-31 (pre-miR-31) can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of pre-miR-31 to investigate the role of distinct structural elements in regulating processing by the Dicer-TRBP complex. We found that the presence or absence of mismatches within the helical stem does not strongly influence Dicer-TRBP processing of the pre-miRNAs. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by the Dicer-TRBP complex. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influences processing by the Dicer-TRBP complex. Our results enrich our understanding of the active role that RNA structure plays in regulating miRNA biogenesis, which has direct implications for the control of gene expression.

Structure of pre-miR-31 reveals an active role in Dicer processing.

As an essential post-transcriptional regulator of gene expression, microRNA (miR) levels must be strictly maintained. The biogenesis of many, but not all, miRs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers and interestingly, its biogenesis is not known to be regulated by protein binding partners. Therefore, the intrinsic structural properties of pre-miR-31 can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of the precursor element of miR-31 (pre-miR-31) to investigate the role of distinct structural elements in regulating Dicer processing. We found that the presence or absence of mismatches within the helical stem do not strongly influence Dicer processing of the pre-miR. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by Dicer. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influence both Dicer binding and processing. Our results enrich our understanding of the active role that RNA structure plays in regulating Dicer processing which has direct implications for control of gene expression.

Solution Structure of NPSL2, A Regulatory Element in the oncomiR-1 RNA.

The miR-17 âˆ¼ 92a polycistron, also known as oncomiR-1, is commonly overexpressed in multiple cancers and has several oncogenic properties. OncomiR-1 encodes six constituent microRNAs (miRs), each enzymatically processed with different efficiencies. However, the structural mechanism that regulates this differential processing remains unclear. Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a structure that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing. Here, we determined the solution structure of NPSL2 using solution NMR spectroscopy. This is the first high-resolution structure of an oncomiR-1 element. NPSL2 adopts a hairpin structure with a large, but highly structured, apical and internal loops. The 10-bp apical loop contains a pH-sensitive A+·C mismatch. Additionally, several adenosines within the apical and internal loops have elevated pKa values. The protonation of these adenosines can stabilize the NPSL2 structure through electrostatic interactions. Our study provides fundamental insights into the secondary and tertiary structure of an important RNA hairpin proposed to regulate miR biogenesis.

pH dependence of C•A, G•A and A•A mismatches in the stem of precursor microRNA-31.

MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A∙A, G∙A, and C∙A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that nucleotides within the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability.

NMR chemical shift assignments of RNA oligonucleotides to expand the RNA chemical shift database.

RNAs play myriad functional and regulatory roles in the cell. Despite their significance, three-dimensional structure elucidation of RNA molecules lags significantly behind that of proteins. NMR-based studies are often rate-limited by the assignment of chemical shifts. Automation of the chemical shift assignment process can greatly facilitate structural studies, however, accurate chemical shift predictions rely on a robust and complete chemical shift database for training. We searched the Biological Magnetic Resonance Data Bank (BMRB) to identify sequences that had no (or limited) chemical shift information. Here, we report the chemical shift assignments for 12 RNA hairpins designed specifically to help populate the BMRB.

Setting priorities for people with intellectual disability/intellectual developmental disorders across the lifespan: a call to action by the World Psychiatric Association.

People with DSM-5 intellectual disability/intellectual developmental disorder (ID/IDD) or ICD-11 disorders of intellectual development (DID) have multiple healthcare needs, but in many countries these needs are neither recognised nor managed effectively. This paper discusses the negative impact that stigma, discrimination and social exclusion have on the identification and care of persons with ID/IDD in low- and middle-income countries (LMICs). It also reviews different models of care for children, adolescents and adults. In discussing some initiatives in LMICs the emphasis is on early diagnosis, with success in providing locally sourced care for affected people and their families. This is where the medical, social and rights-based models of care intersect and is a premise of the person-centred biopsychosocial framework of the World Psychiatric Association's Presidential Action Plan 2020-2023. The plan invites psychiatrists to take a lead in changing the culture of care, as well as medical education, clinical training and research, with a renewed emphasis on workforce integration and service development in terms of community-based rehabilitation strategies.

Single center analysis of patients with H1N1 vaccine-related narcolepsy and sporadic narcolepsy presenting over the same time period.

STUDY OBJECTIVES: We aimed to describe the clinical features of narcolepsy in patients referred to our sleep center between 2009 and 2016, and to compare these features across age groups and between sporadic vs AS03-adjuvanted H1N1 influenza vaccine-related patients. METHODS: This is a retrospective, consecutive study of adult and pediatric narcolepsy patients in the Republic of Ireland. All participants underwent structured assessments, including polysomnography and the Multiple Sleep Latency Test. Brain magnetic resonance imaging, hypocretin levels, and human leukocyte antigen typing were also carried out on the majority of patients. Patients were compared across age groups as well as etiology. RESULTS: The conditions of 40 (74%) patients were vaccine-related. The median age was 13.5 years and time from symptom onset to diagnosis was 112 weeks. Median time from vaccination to symptom onset was 26 weeks. In children, hypnogogic hallucinations and sleep paralysis were less frequent than in adults (17% vs 67%, P = .018 and 0% vs 75%, P < .0005). Sleep latency determined by the Multiple Sleep Latency Test was shorter in children than adults (median 1.75 vs 4 minutes, P = .011). Patients with vaccine-related and sporadic narcolepsies had typical clinical presentations. Vaccine-related patients had longer polysomnography latency (median 10.5 vs 5 minutes, P = .043), longer stage N2 sleep (209.6 ± 44.6 vs 182.3 ± 34.2 minutes, P = .042), and a trend toward longer total sleep times (P = .09). No differences were noted in relation to Multiple Sleep Latency Test, hypocretin, human leukocyte antigen typing, and magnetic resonance imaging. CONCLUSIONS: Results show that vaccine-related patients greatly outnumbered sporadic patients during the study period and suggest that sporadic and vaccine-related narcolepsy are clinically similar entities.

A Tale of Two Transitions: The Unfolding Mechanism of the prfA RNA Thermosensor.

RNA thermosensors (RNATs), found in the 5' untranslated region (UTR) of some bacterial messenger RNAs (mRNAs), control the translation of the downstream gene in a temperature-dependent manner. In Listeria monocytogenes, the expression of a key transcription factor, PrfA, is mediated by an RNAT in its 5' UTR. PrfA functions as a master regulator of virulence in L. monocytogenes, controlling the expression of many virulence factors. The temperature-regulated expression of PrfA by its RNAT element serves as a signal of successful host invasion for the bacteria. Structurally, the prfA RNAT bears little resemblance to known families of RNATs, and prior studies demonstrated that the prfA RNAT is highly responsive over a narrow temperature range. Herein, we have undertaken a comprehensive mutational and thermodynamic analysis to ascertain the molecular determinants of temperature sensitivity. We provide evidence to support the idea that the prfA RNAT unfolding is different from that of cssA, a well-characterized RNAT, suggesting that these RNATs function via distinct mechanisms. Our data show that the unfolding of the prfA RNAT occurs in two distinct events and that the internal loops play an important role in mediating the cooperativity of RNAT unfolding. We further demonstrated that regions distal to the ribosome binding site (RBS) not only contribute to RNAT structural stability but also impact translation of the downstream message. Our collective results provide insight connecting the thermal stability of the prfA RNAT structure, unfolding energetics, and translational control.

Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for the transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes' behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2' hydroxyl acylation, as probed by reverse transcription. Here, reanalysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on (A)12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3' untranslated region requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger an unexpectedly effective reverse transcriptase bypass and that chemical modifications in mRNA poly(A) tails may be generally undercounted.

Advanced approaches for elucidating structures of large RNAs using NMR spectroscopy and complementary methods.

NMR spectroscopy is a key technique that has significantly advanced our understanding of RNA structure and dynamics. However, determination of large RNA structures by NMR spectroscopy remains a significant technical challenge. In this review, we highlight advances that facilitate NMR studies of large RNAs, including methods for sample preparation, isotope labeling strategies, and data acquisition. In addition, we review hybrid approaches that have been instrumental in the structure determination of large RNAs.

Structure of the zinc-finger antiviral protein in complex with RNA reveals a mechanism for selective targeting of CG-rich viral sequences.

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

Advances that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy.

The characterization of functional yet nonprotein coding (nc) RNAs has expanded the role of RNA in the cell from a passive player in the central dogma of molecular biology to an active regulator of gene expression. The misregulation of ncRNA function has been linked with a variety of diseases and disorders ranging from cancers to neurodegeneration. However, a detailed molecular understanding of how ncRNAs function has been limited; due, in part, to the difficulties associated with obtaining high-resolution structures of large RNAs. Tertiary structure determination of RNA as a whole is hampered by various technical challenges, all of which are exacerbated as the size of the RNA increases. Namely, RNAs tend to be highly flexible and dynamic molecules, which are difficult to crystallize. Biomolecular nuclear magnetic resonance (NMR) spectroscopy offers a viable alternative to determining the structure of large RNA molecules that do not readily crystallize, but is itself hindered by some technical limitations. Recently, a series of advancements have allowed the biomolecular NMR field to overcome, at least in part, some of these limitations. These advances include improvements in sample preparation strategies as well as methodological improvements. Together, these innovations pave the way for the study of ever larger RNA molecules that have important biological function. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Are patterns of violence and aggression at presentation in patients with first-episode psychosis temporally stable? A comparison of 2 cohorts.

AIM: The aim of this study is to assess the prevalence and clinical correlates of aggression and violence in individuals presenting with first-episode psychosis (FEP) and to evaluate whether this prevalence has changed in recent years when compared to a similar previous study. METHODS: Retrospective cross-sectional study of clinical case notes and database records using a keyword search of a sample of patients with FEP (n = 132) aged between 18 and 65 years presenting from a geographically defined catchment area to a secondary referral psychiatric service over a 4-year period (2010-2013 inclusive). Use of the Modified Overt Aggression Scale to retrospectively assess aggression and violence in the week prior to, and post, presentation with FEP. RESULTS: The overall proportion of individuals found to be aggressive and violent was 36% and 29%, respectively. These rates were similar to the 1995 to 1998 cohort (ie, 33% and 29%). A higher percentage of our sample (22%) was violent in the week prior to presentation compared to the 1995 to 1998 cohort (13%). Aggression was independently associated with involuntary (odds ratio [OR] = 4.085, 95% confidence interval [CI] 1.310-12.733) and inpatient treatment status (OR = 0.109, 95% CI 0.023-0.532) in the week prior to presentation and with high activation (OR = 6.770, 95% CI 1.372-33.394) and involuntary treatment status (OR = 10.163, 95% CI 2.257-45.759) in the week following presentation. Violence was associated with involuntary (OR = 3.691, 95% CI 1.197-11.382) and inpatient status (OR = 0.096, 95% CI 0.020-0.465) in the week prior to and with high activation (OR = 29.513 95% CI 1.879-463.676) the week following presentation. CONCLUSIONS: Aggression and violence rates in FEP appear relatively stable over time.

Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach.

Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]2; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.

NMR Studies of the Structure and Function of the HIV-1 5'-Leader.

The 5'-leader of the human immunodeficiency virus type 1 (HIV-1) genome plays several critical roles during viral replication, including differentially establishing mRNA versus genomic RNA (gRNA) fates. As observed for proteins, the function of the RNA is tightly regulated by its structure, and a common paradigm has been that genome function is temporally modulated by structural changes in the 5'-leader. Over the past 30 years, combinations of nucleotide reactivity mapping experiments with biochemistry, mutagenesis, and phylogenetic studies have provided clues regarding the secondary structures of stretches of residues within the leader that adopt functionally discrete domains. More recently, nuclear magnetic resonance (NMR) spectroscopy approaches have been developed that enable direct detection of intra- and inter-molecular interactions within the intact leader, providing detailed insights into the structural determinants and mechanisms that regulate HIV-1 genome packaging and function.

NMR detection of intermolecular interaction sites in the dimeric 5'-leader of the HIV-1 genome.

HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5'-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5'-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5' (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies.

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5' untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

RNA structure. Structure of the HIV-1 RNA packaging signal.

The 5' leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a (2)H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; Ψ(CES)). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics.

Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg(2+) cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg(2+), mimicking the chelation by the ß- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg(2+) ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg(2+)-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.