Sheena Saayman

The biogenesis and characterization of mammalian microRNAs of mirtron origin

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT–PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.

Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex

HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a “hotspot” for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a “functional cure” of HIV/AIDS.

Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors

Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3′ position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi.

The therapeutic application of CRISPR/Cas9 technologies for HIV

Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system. Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

The emerging role of long non-coding RNAs in HIV infection.

The discovery of long non-coding RNAs (lncRNAs) and the elucidation of the mechanisms by which they affect different disease states are providing researchers with a better understanding of a wide array of disease pathways. Moreover, lncRNAs are presenting themselves as both unique diagnostic biomarkers as well as novel targets against which to develop new therapeutics. Here we will explore the intricate network of non-coding RNAs associated with infection by the human immunodeficiency virus (HIV). Non-coding RNAs derived from both the human host as well as those from HIV itself are emerging as important regulatory elements. We discuss here the various mechanisms through which both small and long non-coding RNAs impact viral replication, pathogenesis and disease progression. Given the lack of an effective vaccine or cure for HIV and the scale of the current pandemic, a deeper understanding of the complex interplay between non-coding RNAs and HIV will support the development of innovative strategies for the treatment of HIV/acquired immunodeficiency disease (AIDS).

MYCNOS functions as an antisense RNA regulating MYCN.

Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci.

Effective Pol III-Expressed Long Hairpin RNAs Targeted to Multiple Unique Sites of HIV-1

The RNA interference (RNAi) pathway has in recent years been exploited for the development of novel antiviral therapies. The emergence of viral escape mutants, however, is a major impediment to the use of RNAi effectors to treat highly mutable viruses such as HIV-1. A combinatorial approach is therefore required for long-term inhibition of gene expression. RNA Pol III-driven long hairpin RNA (lhRNA) duplexes can be cleaved several times by Dicer, yielding multiple functional siRNAs from a single construct. Here we describe a method for the generation of ectopically expressed U6-lhRNAs encoding three separate siRNA sequences targeting unique sites in HIV-1. This methodological overview explains some crucial aspects of lhRNA design and cloning as well as facile experiments to determine their efficacy in cell culture.

Long Non-coding RNA BGas Regulates the Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic fibrosis (CF) is a life-shortening genetic disease. The root cause of CF is heritable recessive mutations that affect the cystic fibrosis transmembrance conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface. We show that CFTR is regulated transcriptionally by the actions of a novel long noncoding RNA (lncRNA), designated as BGas, that emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein coding sense strand. We find that BGas functions in concert with several proteins including HMGA1, HMGB1, and WIBG to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene and thereby affects transcription. Suppression of BGas or its associated proteins results in a gain of both CFTR expression and chloride ion function. The observations described here highlight a previously underappreciated mechanism of transcriptional control and suggest that BGas may serve as a therapeutic target for specifically activating expression of CFTR.

HIV Latency and the Noncoding RNA Therapeutic Landscape

The Human Immunodeficiency Virus (HIV) belongs to the subfamily of lentiviruses that are characterized by long incubation periods and chronic, persistent infection. The virus integrates into the genome of infected CD4+ cells and, in a subpopulation of cells, adopts a transcriptionally silent state, a process referred to a viral latency. This property makes it exceedingly difficult to therapeutically target the virus and eradicate infection. If left untreated, the inexorable demise of the infected individuals immune system ensues, a causal result of Acquired Immunodeficiency Syndrome (AIDS). Latently infected cells provide a reservoir that maintains viral infection indefinitely. In this chapter we explore the role of noncoding RNAs in HIV infection and in the establishment and maintenance of viral latency. Both short and long noncoding RNAs are endogenous modulators of epigenetic regulation in human cells and play an active role in gene expression. Lastly, we explore therapeutic modalities based on expressed RNAs that are capable of countering infection, transcriptionally regulating the virus, and suppressing or activating the latent state.