Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

Tan, Xu; Lu, Zhi John; Gao, Geng; Xu, Qikai; Hu, Long; Fellmann, Christof; Li, Mamie Z.; Qu, Hongjing; Lowe, Scott W.; Hannon, Gregory J.; Elledge, Stephen J.

doi:10.1073/pnas.1119873109

Cited by 27 publications

(29 citation statements)

References 29 publications

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“…On the other hand, this laboratory has reported that miRNA binding to HIV-1 transcripts, while detectable, is ~100-fold less efficient than miRNA binding to cellular mRNAs expressed contemporaneously in HIV-1-infected T cells (5). This finding is consistent with data demonstrating that the HIV-1 RNA genome is highly structured (6) and that RNA secondary structure inhibits miRNA binding, including to predicted miRNA binding sites present on HIV-1 transcripts (7–9). Moreover, we recently demonstrated that mutational inactivation of human Dicer, which blocks the production of all cellular miRNAs, does not enhance HIV-1 replication or gene expression (10), thus strongly suggesting that HIV-1 has indeed evolved to avoid inhibition by cellular miRNAs.…”

Section: Introductionsupporting

confidence: 91%

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

Bogerd

Kennedy

Whisnant

et al. 2017

mBio

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Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.

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Section: Introductionsupporting

confidence: 91%

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

Bogerd

Kennedy

Whisnant

et al. 2017

mBio

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“…In viral genomes, the experimentally determined secondary structure of the target site is strongly anticorrelated with shRNA activity (23). However, we found no strong predictive power of either the thermodynamic stability of the mRNA segment containing the target site or of the accessibility of the target of the seed sequence only as predicted by the unafold algorithm (24) (Fig.…”

Section: Resultscontrasting

confidence: 39%

Next-generation libraries for robust RNA interference-based genome-wide screens

Kampmann

Horlbeck

Chen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.functional genomics | shRNA | genetic screen | pooled screen | microRNA F unctional genomics approaches in mammalian cells have the potential to dissect gene functions and to complement observational genomics approaches for the identification of disease mechanisms and therapeutic strategies. For many years, RNA interference (RNAi) was the technology of choice for loss-offunction screens in mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches recently developed by us and others (1-4) provide a highly promising orthogonal strategy. In particular, CRISPR interference (CRISPRi) has reduced off-target effects and can reach high levels (90-100%) of knockdown (1), and CRISPR cutting can generate null alleles.Despite the advantages of CRISPR-based strategies, there are still important uses for RNAi technology. RNAi is a singlecomponent system that works in otherwise unengineered cells, and can thus be used in challenging biological contexts. Because CRISPRi blocks transcription at the transcription start site of endogenous genes, it does not allow selective targeting of functionally distinct coding and noncoding RNAs derived from the same primary transcript, such as splice isoforms or noncoding RNAs embedded in the introns of coding transcripts, whereas RNAi reagents can be designed for specific targeting of mature RNAs (mRNAs).Hence, there is a continued need to improve RNAi-based screening platforms. We have previously established a quantitative framework to derive robust results from pooled screens of ultracomplex short-hairpin RNA (shRNA) libraries that target each gene with ∼25 independent shRNAs and contain thousands of negative-control shRNAs (5). Such complex libraries can be constructed using massively parallel oligonucleotide synthesis, and phenotypes in pooled populations can be determined using n...

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“…Although the functions of endogenous mRNA pseudouridylation events are not yet known, the established effects of Ψ on RNA structure suggest many possibilities for posttranscriptional regulation through the impact of mRNA structure on translation initiation efficiency, ribosome pausing, RNA localization, and regulation by RNA interference ( Jambhekar & DeRisi, 2007;Kudla, Murray, Tollervey, & Plotkin, 2009;Shah, Ding, Niemczyk, Kudla, & Plotkin, 2013;Somogyi, Jenner, Brierley, & Inglis, 1993;Tan et al, 2012). Furthermore, mRNA pseudouridylation may provide a mechanism for dynamically altering the genetic code.…”

Section: Rna Modificationsmentioning

confidence: 97%

Pseudo-Seq

Carlile

Rojas-Durán

Gilbert

2015

Methods in Enzymology

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RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.

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Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

Cited by 27 publications

References 29 publications

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

Next-generation libraries for robust RNA interference-based genome-wide screens

Pseudo-Seq

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