C911: A Bench-Level Control for Sequence Specific siRNA Off-Target Effects

<div><p>Small interfering RNAs (siRNAs) have become a ubiquitous experimental tool for down-regulating mRNAs. Unfortunately, off-target effects are a significant source of false positives in siRNA experiments and an effective control for them has not previously been identified. We introduce two methods of mismatched siRNA design for negative controls based on changing bases in the middle of the siRNA to their complement bases. To test these controls, a test set of 20 highly active siRNAs (10 true positives and 10 false positives) was identified from a genome-wide screen performed in a cell-line expressing a simple, constitutively expressed luciferase reporter. Three controls were then synthesized for each of these 20 siRNAs, the first two using the proposed mismatch design methods and the third being a simple random permutation of the sequence (scrambled siRNA). When tested in the original assay, the scrambled siRNAs showed significantly reduced activity in comparison to the original siRNAs, regardless of whether they had been identified as true or false positives, indicating that they have little utility as experimental controls. In contrast, one of the proposed mismatch design methods, dubbed C911 because bases 9 through 11 of the siRNA are replaced with their complement, was able to completely distinguish between the two groups. False positives due to off-target effects maintained most of their activity when the C911 mismatch control was tested, whereas true positives whose phenotype was due to on-target effects lost most or all of their activity when the C911 mismatch was tested. The ability of control siRNAs to distinguish between true and false positives, if widely adopted, could reduce erroneous results being reported in the literature and save research dollars spent on expensive follow-up experiments.</p></div

siRNAs Selected as True and False Positives.

<p>siRNAs Selected as True and False Positives.</p

On and off-target effects of siRNAs and their controls.

<p>An siRNA (<b>A, left panel</b>) consisting of two complementary 19-mers of RNA (with two-base overhangs) is divided here conceptually into the 5′ end of the anti-sense strand (teal) the middle of the siRNA (black) and the 3′ end of the anti-sense strand (red). siRNAs are designed to be the reverse-completment of the mRNA sequence they are targeted to down-regulate (<b>A</b>, <b>middle panel</b>), but matches of the seed sequence of an siRNA to the 3′UTR of other mRNAs can result in their off-target down-regulation as well (<b>A</b>, <b>right panel</b>). A scrambled siRNA (<b>B</b>) eliminates the match to the target mRNA and thus will not down-regulate it, but also eliminates the off-target effects due to matches to the seed sequence (while, perhaps, creating new off-target effects against the new seed sequence). The C911 mismatch control (<b>C</b>) reduces or eliminates the down-regulation of the targeted mRNA by taking the complement of the middle three bases (green), but maintains the off-target effects of the original siRNA by keeping anti-sense and sense strand seed sequences intact. In this manner, comparison of effects elicited by the original siRNA and the C911 mismatch control should allow us to distinguish phenotypes that are due to down-regulation of the intended target rather than off-target effects.</p

Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP)

<div><p>The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.</p></div

CRISPR targeting of <i>TRIM25</i> leads to increased virus replication and both the RING and CCD domains of TRIM25 are required for ZAP activation.

<p><b>(A)</b> Wild type (clone E) and TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells (clones D and F) were transfected with empty vector or vector expressing ZAPS or ZAPL and infected with Toto1101/Luc (MOI = 0.01) 2 days post-transfection. <b>(B)</b> TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells (clones D and F) were reconstituted with expression of FL or truncated TRIM25 (ΔRING, ΔCCD) and/or ZAPS or ZAPL, and infected with Toto1101/Luc (MOI = 10) 2 days post-transfection. <b>(A and B)</b> The data is representative of 2 independent experiments performed on both clones D and F. Cell lysates were harvested for measurement of luciferase activity at 24 h p.i. Relative luciferase units represent the level of SINV replication. Asterisks indicate statistically significant differences (Student’s t-test, *, p<0.05; **, p<0.005; ***, p<0.0005).</p

Both ZAPS and ZAPL are ubiquitinated by TRIM25.

<p>Cells were lysed in denaturing conditions to ensure pulldown of ZAP only and not ZAP-associated proteins. <b>(A)</b> WCL of 293T cells transfected with vector expressing HA-tagged ubiquitin (Ub), and mock infected or infected with the SINV Toto1101 strain (MOI = 1) for 18 hours were used for immunoprecipitation of endogenous ZAP with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(B)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing HA-tagged Ub, and ZAPS or ZAPL were used for immunoprecipitation of overexpressed ZAP with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(C)</b> WCL of wild type and TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing HA-tagged Ub, and ZAPS or ZAPL were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The data is representative of 2 independent experiments performed on both clones D and F. Only data for clone D is shown here. <b>(D)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of endogenous Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(E)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vector expressing HA-tagged Ub, ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 2 independent experiments. <b>(F)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vector expressing HA-tagged wild type (WT), K48 or K63 Ub, ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of HA-tagged WT or mutant Ub in the ZAP pulldown is shown. The data is representative of 2 independent experiments.</p

Secondary screen confirms the ZAP-dependent and -independent antiviral effects of hits.

<p>A customized library of individual siRNAs (Ambion) targeting 102 hits from the primary screen was tested in triplicate in two different cell lines. Distribution of average Z scores for each of the 3 siRNAs targeting a candidate gene in the secondary screen is shown here. The screen was performed with <b>(A)</b> 6.25 nM or <b>(B)</b> 25 nM individual siRNAs in 293 cells induced to express ZAPS (T-REx-hZAP), and with <b>(C)</b> 6.25 nM or <b>(D)</b> 25 nM individual siRNAs in 293 cells induced to express the rZAPC88R dominant negative mutant (T-REx-rZAPC88R). Each dot represents the average Z score of an individual siRNA tested in triplicate. Silenced genes with an average Z score of >3 for at least 2 out of 3 siRNAs are identified and the siRNAs are labeled in color. <b>(C and D)</b> The average Z scores of <i>TRIM25</i> and <i>ZC3HAV1</i> are also plotted to indicate that TRIM25 does not have ZAP-independent antiviral effects.</p

TRIM25 synergizes with ZAP to block SINV replication.

<p><b>(A)</b> Candidate genes that significantly increased SINV replication in the secondary screen when silenced by individual siRNAs at both concentrations were validated in a larger scale 24-well plate format. Triplicate wells of T-REx-hZAP cells were transfected with the indicated siRNA, induced to express ZAPS, and infected with SINV Toto1101/Luc at a MOI of 10. Each symbol represents the value obtained from a single well after 24 h of infection. White circles represent results using pooled siRNA controls that were either NT or <i>ZC3HAV1</i>-specific. The data is representative of 3 independent experiments. Asterisks indicate statistically significant differences (Student’s t-test, **, p<0.005; ***, p<0.0005; ****, p<0.0001). <b>(B)</b> Triplicate wells of T-REx-hZAP cells were transfected with the indicated siRNA, induced to express ZAPS, and infected with SINV Toto1101/Luc at a MOI of 10. Protein expression levels of TRIM25 and ZAP for the same transfections in a duplicate well were determined by immunoblotting. β-actin was used as a loading control. The data is representative of 4 independent experiments. The p-value from Student’s t-test is shown. <b>(C)</b> SINV replication in infected 293T cells in which <i>ZC3HAV1</i> (left) or <i>TRIM25</i> (middle) were silenced, and in <i>ZC3HAV1</i>-null 293T cells in which <i>TRIM25</i> was silenced (right) is plotted. At 48 h post-transfection with siRNA, cells were infected with SINV Toto1101/Luc at a MOI of 0.01, and lysed at 6, 12, 24, and 40 h p.i. for measurement of luciferase activity. The data is representative of 3 independent experiments. Asterisks indicate statistically significant differences (two-way ANOVA, *, p<0.05; **, p<0.01; ****, p<0.0001).</p

A loss-of-function RNAi screen uncovers many genes that significantly reduce the antiviral activity of ZAP when silenced.

<p><b>(A)</b> The experimental outline of the genome-wide siRNA screen is shown. T-REx-hZAP cells transfected with control or gene-specific siRNA were treated with doxycycline to induce ZAPS overexpression one day post-transfection and infected with SINV Toto1101/Luc two days post-transfection. Cell lysates were harvested for measurement of luciferase activity at 24 h post-infection (p.i.). Relative luciferase units represent the level of SINV replication. Cells treated with the control non-targeting (NT) pooled siRNA have low SINV replication while ZAP knockdown by <i>ZC3HAV1</i>-specific pooled siRNA rescues viral replication by 2 logs. The large dynamic range in which hypothetical hits (ZAP cofactors) were identified is plotted on the right side of the graph. <b>(B)</b> Pooled siRNAs targeting the entire human genome (Dharmacon) were tested in triplicate and genes with an average robust Z score of greater than 3 are plotted. <i>ZC3HAV1</i> is highlighted in red while the top hits immediately following <i>ZC3HAV1</i> are highlighted in blue.</p