Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity

The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5'-C, antisense strand: 3'-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.

Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3

Recent studies have shown that long noncoding RNAs (lncRNAs) are critical regulators in the central nervous system (CNS). However, their roles in the cerebellum are currently unclear. In this work, we identified the isoform 204 of lncRNA Gm2694 (designated as lncRNA-Promoting Methylation (lncRNA-PM)) is highly expressed in the cerebellum and derived from the antisense strand of the upstream region of Cerebellin-1 (Cbln1), a well-known critical cerebellar synaptic organizer. LncRNA-PM exhibits similar spatiotemporal expression pattern as Cbln1 in the postnatal mouse cerebellum and activates the transcription of Cbln1 through Pax6/Mll1-mediated H3K4me3. In mouse cerebellum, lncRNA-PM, Pax6/Mll1, and H3K4me3 are all associated with the regulatory regions of Cbln1. Knockdown of lncRNA-PM in cerebellum causes deficiencies in Cbln1 expression, cerebellar synaptic integrity, and motor function. Together, our work reveals an lncRNA-mediated transcriptional activation of Cbln1 through Pax6-Mll1-H3K4me3 and provides novel insights of the essential roles of lncRNA in the cerebellum.

Identification of a Long Non-Coding RNA Derived From the Antisense Strand of the Thyroid Hormone Receptor Beta Gene

Background: Long non-coding RNAs (lncRNAs) have various functions. Here, we describe a novel lncRNA which is induced from the antisense strand of the thyroid hormone receptor beta (THRB) gene. Methods: We sequenced RNA from leukocytes and identified a novel lncRNA derived from the antisense strand of the THRB gene. The lncRNA was named THRB antisense RNA 2 (THRB-AS2). Transcriptional regulation by THRB-AS2 was assessed by reporter assays. The putative translation initiation codons of THRB-AS2 were mutated to assess potential THRB-AS2 protein function. Results: THRB-AS2 stimulated the activities of multiple promoters irrespective of thyroid hormone response elements. Thyroid hormone treatment did not affect the function of THRB-AS2. These data suggest that THRB-AS2 is a potential general transcriptional activator and has broad specificity on target genes. Mutations in putative translation initiation codons of THRB-AS2 did not affect function, suggesting that THRB-AS2 acts as an RNA molecule. Conclusions: Although a small number of lncRNAs derived from antisense strand of the THRB gene, such as THRB-AS1, has been already reported, their functions remain largely unknown. To our knowledge, this is the first report of the functions of a lncRNA derived from the antisense strand of the THRB gene.

Borf: Improved ORF prediction in de-novo assembled transcriptome annotation

ABSTRACTORF prediction in de-novo assembled transcriptomes is a critical step for RNA-Seq analysis and transcriptome annotation. However, current approaches do not appropriately account for factors such as strand-specificity and incompletely assembled transcripts. Strand-specific RNA-Seq libraries should produce assembled transcripts in the correct orientation, and therefore ORFs should only be annotated on the sense strand. Additionally, start site selection is more complex than appreciated as sequences upstream of the first start codon need to be correctly annotated as 5’ UTR in completely assembled transcripts, or part of the main ORF in incomplete transcripts. Both of these factors influence the accurate annotation of ORFs and therefore the transcriptome as a whole. We generated four de-novo transcriptome assemblies of well annotated species as a gold-standard dataset to test the impact strand specificity and start site selection have on ORF prediction in real data. Our results show that prediction of ORFs on the antisense strand in data from stranded RNA libraries results in false-positive ORFs with no or very low similarity to known proteins. In addition, we found that up to 23% of assembled transcripts had no stop codon upstream and in-frame of the first start codon, instead comprising a sequence of upstream codons. We found the optimal length cutoff of these upstream sequences to accurately classify these transcripts as either complete (upstream sequence is 5’ UTR) or 5’ incomplete (transcript is incompletely assembled and upstream sequence is part of the ORF). Here, we present Borf, the better ORF finder, specifically designed to minimise false-positive ORF prediction in stranded RNA-Seq data and improve annotation of ORF start-site prediction accuracy. Borf is written in Python3 and freely available at https://github.com/betsig/borf.

Over-representation of MEF-2 isoforms (A/C) is associated with HTLV-1-induced acute ATLL and their recruitment to 3’LTR facilitates HBZ expression from the antisense promoter via interactions with Menin and Jun D

Background. HTLV-1 is a complex human retrovirus and an etiologic agent causing a malignant and intractable T-cell neoplasia termed Adult T-cell leukemia and lymphoma (ATLL). Patients suffering from ATLL present with poor prognoses and a dearth of treatment options warranting a continuous need to develop novel therapeutic targets. In contrast to the HTLV-1 transactivator protein Tax, HTLV-1 bZIP protein (HBZ) maintains its expression in ATLL cells. The HBZ gene is encoded from the antisense strand of the provirus and is not under the transcriptional control of the 5’ long terminal repeat (LTR) unlike other viral genes such as Tax. Few modifications have been reported in the 3’LTR, which regulates HBZ expression. Herein, we delineate the activities of a transcription factor MEF (Myocyte enhancer factor)-2 at both 5’ and 3’LTRs in the context of ATLL progression and maintenance. Results. In this study, we report that two MEF isoforms (2A and 2C) are highly overexpressed in acute ATLL patients from North America. These isoforms are recruited to the viral promoters at both the 5’ and 3’LTRs. Their knockdown by shRNAs resulted in the downregulation of Tax and HBZ expression as well as a significant decrease in proliferation and cell cycle arrest in ATLL cells. Similarly, chemical inhibition of MEF proteins by MC1568 (a selective Class IIa HDAC inhibitor) resulted in the cytotoxicity of ATLL cells in vitro as well as reduction of proviral load and viral gene expression in vivo. At the molecular level, high enrichment of MEF-2C occurred at the 3’LTR along with cofactors Menin, Jun D, and Sp1/Sp3 thus providing a novel mechanism of regulation at the antisense promoter of HTLV-1. Conclusions. This study establishes MEF-2 as critical players in ATLL, which interacts with Tax and HBZ at their respective promoters highlighting a novel mechanism of regulation at the 3’LTR involving Jun D and Menin. MEF signaling represent a potential target for therapeutic intervention.

lncSAMM50 Enhances Adipogenic Differentiation of Buffalo Adipocytes With No Effect on Its Host Gene

Fat deposition is one of the most important traits that are mediated by a set of complex regulatory factors in meat animals. Several researches have revealed the significant role of long non-coding RNAs (lncRNAs) in fat deposition while the precise regulatory mechanism is still largely elusive. In this study, we investigated the lncRNA profiles of adipose and muscle tissues in buffalo by using the Illumina HiSeq 3000 platform. In total, 43,809 lncRNAs were finally identified based on the computer algorithm. A comparison analysis revealed 241 lncRNAs that are differentially expressed (DE) in adipose and muscle tissues. We focused on lncSAMM50, a DE lncRNA that has a high expression in adipose tissue. Sequence alignment showed that lncSAMM50 is transcribed from the antisense strand of the upstream region of sorting and assembly machinery component 50 homolog (SAMM50), a gene involved in the function of mitochondrion and is subsequently demonstrated to inhibit the adipogenic differentiation of 3T3-L1 adipocyte cells in this study. lncSAMM50 is highly expressed in adipose tissue and upregulated in the mature adipocytes and mainly exists in the nucleus. Gain-of-function experiments demonstrated that lncSAMM50 promotes the adipogenic differentiation by upregulating adipogenic markers but with no effect on its host gene SAMM50 in buffalo adipocytes. These results indicate that lncSAMM50 enhances fat deposition in buffalo and provide a new factor for the regulatory network of adipogenesis.

Emerging Perspectives on Dipeptide Repeat Proteins in C9ORF72 ALS/FTD

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide expansion in the chromosome 9 open reading frame 72 gene (C9ORF72). This hexanucleotide expansion consists of GGGGCC (G4C2) repeats that have been implicated to lead to three main modes of disease pathology: loss of function of the C9ORF72 protein, the generation of RNA foci, and the production of dipeptide repeat proteins (DPRs) through repeat-associated non-AUG (RAN) translation. Five different DPRs are currently known to be formed: glycine–alanine (GA) and glycine–arginine (GR) from the sense strand, proline–alanine (PA), and proline–arginine (PR) from the antisense strand, and glycine–proline (GP) from both strands. The exact contribution of each DPR to disease pathology is currently under intense scrutiny and is still poorly understood. However, recent advances in both neuropathological and cellular studies have provided us with clues enabling us to better understand the effect of individual DPRs on disease pathogenesis. In this review, we compile the current knowledge of specific DPR involvement on disease development and highlight recent advances, such as the impact of arginine-rich DPRs on nucleolar protein quality control, the correlation of poly-GR with neurodegeneration, and the possible involvement of chimeric DPR species. Further, we discuss recent findings regarding the mechanisms of RAN translation, its modulators, and other promising therapeutic options.