Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

Petri, Sebastian; Dueck, Anne; Lehmann, Gerhard; Putz, Nicholas; Rüdel, Sabine; Kremmer, Elisabeth; Meister, Gunter

doi:10.1261/rna.2348111

Cited by 64 publications

(65 citation statements)

References 45 publications

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“…5A). Of note, the amount of pulled down Ago proteins resembles the protein expression levels (29,30). For further affinity measurements, we tested possible protein aggregation using dynamic light scattering (DLS) (Materials and Methods and Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

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

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100

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MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.

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Section: Resultsmentioning

confidence: 99%

Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

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

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100

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“…Annotation of Argonaute-related literature yielded a number of reposts describing covalent modifications of AGO proteins implicated in post-translational regulations, namely phosphorylation (Zeng et al, 2008;Rudel et al, 2011;Horman et al, 2013;Mazumder et al, 2013;Shen et al, 2013;Lopez-Orozco et al, 2015;Patranabis andBhattacharyya, 2016), prolyl 4-hydroxylation (Qi et al, 2008;Wu et al, 2011), sumoylation (Josa-Prado et al, 2015, ubiquitination (Rybak et al, 2009; The present document has been produced and adopted by the bodies identified above as authors. This task has been carried out exclusively by the authors in the context of a contract between the European Food Safety Authority and the authors, awarded following a tender procedure.…”

Section: Post-translational Modifications Of Ago Proteinsmentioning

confidence: 99%

“…Phosphorylation has been detected on AGO1 and AGO2 in the PAZ domain (pS253, pT303, pT307), the PIWI domain (pS798), in the L2 linker region (pS387, pY393) and in the MID domain (pY529) (Rudel et al, 2011).…”

Section: Phosphorylationmentioning

confidence: 99%

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Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

Pačes

Nič²,

Novotný³

et al. 2017

EFS3

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This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on~1,400 cumulative references chosen from~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter.

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“…2 D and E). Relative Ago protein levels have only been estimated before (21,22). Such earlier quantifications relied on antibodies with different affinities and thus might not be accurate.…”

Section: Specific Isolation and Characterization Of Ago Protein Complmentioning

confidence: 99%

Biochemical isolation of Argonaute protein complexes by Ago-APP

Hauptmann

Schraivogel

Bruckmann

et al. 2015

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

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121

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During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.

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Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

Cited by 64 publications

References 45 publications

Structural features of Argonaute–GW182 protein interactions

Structural features of Argonaute–GW182 protein interactions

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

Biochemical isolation of Argonaute protein complexes by Ago-APP

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