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Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

View Article: PubMed Central - PubMed

ABSTRACT

Background: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.

Results: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.

Conclusions: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

No MeSH data available.


Related in: MedlinePlus

NTC siRNAs have differential effects on L1 reporter assays. a Wells show G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of no siRNA (mock, with transfection reagent only) or 10 nM NTC #3 siRNA. Graph at right shows EGFP fluorescence from cells pretreated with 10 nM NTC #3 siRNA or mock (M); results from duplicate wells are shown. bTop row shows G418-resistant colonies resulting from the transfection of the L1 reporter JM101 in the presence or absence of 25 nM of indicated siRNA; bottom row shows effect of 25 nM of indicated siRNA on cell growth. c Mean Fluc (left) and Rluc (second from right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter pYX017 in the presence of no siRNA (M) or 25 nM NTC #3 or NTC #5; averages were derived from data shown in (d) by first averaging technical replicates for each biological sample (n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3; average Fluc/Rluc ratios (third from right) are also shown. d Individual luminescence values are shown for Fluc (blue) and Rluc (red) obtained from lysates of HeLa cells transfected with pYX017 and the indicated siRNA; technical replicates are side-by-side; biological replicates are indicated with subscripts
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Fig6: NTC siRNAs have differential effects on L1 reporter assays. a Wells show G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of no siRNA (mock, with transfection reagent only) or 10 nM NTC #3 siRNA. Graph at right shows EGFP fluorescence from cells pretreated with 10 nM NTC #3 siRNA or mock (M); results from duplicate wells are shown. bTop row shows G418-resistant colonies resulting from the transfection of the L1 reporter JM101 in the presence or absence of 25 nM of indicated siRNA; bottom row shows effect of 25 nM of indicated siRNA on cell growth. c Mean Fluc (left) and Rluc (second from right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter pYX017 in the presence of no siRNA (M) or 25 nM NTC #3 or NTC #5; averages were derived from data shown in (d) by first averaging technical replicates for each biological sample (n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3; average Fluc/Rluc ratios (third from right) are also shown. d Individual luminescence values are shown for Fluc (blue) and Rluc (red) obtained from lysates of HeLa cells transfected with pYX017 and the indicated siRNA; technical replicates are side-by-side; biological replicates are indicated with subscripts

Mentions: While investigating the effect of p38δ on L1 retrotransposition, we performed siRNA experiments using a SMARTpool mixture against p38δ (Dharmacon, M-003591-02-0005) and the NTC siRNA #3 (Dharmacon). Although the siRNA against p38δ dramatically reduced the number of G418-resistant colonies relative to NTC #3, RT-PCR showed no significant knockdown of the p38δ transcript (data not shown). Interestingly, however, NTC #3 considerably increased colony density relative to the mock control (Fig. 6a left). EGFP fluorescence from cells pretreated with siRNA prior to transfection suggested that the siRNA had little impact on transfection efficiency (Fig. 6a right). Given these unexpected results, we tested an additional control siRNA, NTC #5, also from Dharmacon. In marked contrast to NTC #3, NTC #5 dramatically reduced G418-resistant colonies relative to the mock control (Fig. 6b top). Neither NTC dramatically affected cell growth, though NTC #3 had a slight inhibitory effect (Fig. 6b bottom). It is notable that unlike p38δ-WT, the NTC siRNAs exerted their respective effects similarly on both Fluc luminescence and G418-resistant colony formation (Fig. 6b top, c left and d). However, L1 activity as reported by the Fluc/Rluc ratio appears to be decreased by NTC #3 rather than increased (Fig. 6c). We did not further investigate potential causes for these results. Information on Dharmacon’s website states that each NTC is reported to contain a minimum of 4 mismatches to all human, mouse and rat genes and to have minimal effects in genome-wide targeting via microarray analyses. We did not test Dharmacon’s NTC #1, as it was reported to increase cell growth (personal communication, Dharmacon), nor NTC #2 or #4 due to their targeting of Firefly luciferase (Dharmacon website).Fig. 6


Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
NTC siRNAs have differential effects on L1 reporter assays. a Wells show G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of no siRNA (mock, with transfection reagent only) or 10 nM NTC #3 siRNA. Graph at right shows EGFP fluorescence from cells pretreated with 10 nM NTC #3 siRNA or mock (M); results from duplicate wells are shown. bTop row shows G418-resistant colonies resulting from the transfection of the L1 reporter JM101 in the presence or absence of 25 nM of indicated siRNA; bottom row shows effect of 25 nM of indicated siRNA on cell growth. c Mean Fluc (left) and Rluc (second from right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter pYX017 in the presence of no siRNA (M) or 25 nM NTC #3 or NTC #5; averages were derived from data shown in (d) by first averaging technical replicates for each biological sample (n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3; average Fluc/Rluc ratios (third from right) are also shown. d Individual luminescence values are shown for Fluc (blue) and Rluc (red) obtained from lysates of HeLa cells transfected with pYX017 and the indicated siRNA; technical replicates are side-by-side; biological replicates are indicated with subscripts
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Fig6: NTC siRNAs have differential effects on L1 reporter assays. a Wells show G418-resistant colonies resulting from transfection of the L1 reporter JM101 in the presence of no siRNA (mock, with transfection reagent only) or 10 nM NTC #3 siRNA. Graph at right shows EGFP fluorescence from cells pretreated with 10 nM NTC #3 siRNA or mock (M); results from duplicate wells are shown. bTop row shows G418-resistant colonies resulting from the transfection of the L1 reporter JM101 in the presence or absence of 25 nM of indicated siRNA; bottom row shows effect of 25 nM of indicated siRNA on cell growth. c Mean Fluc (left) and Rluc (second from right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter pYX017 in the presence of no siRNA (M) or 25 nM NTC #3 or NTC #5; averages were derived from data shown in (d) by first averaging technical replicates for each biological sample (n = 2), then using this value to average biological replicates; error bars represent SEM of biological samples, n = 3; average Fluc/Rluc ratios (third from right) are also shown. d Individual luminescence values are shown for Fluc (blue) and Rluc (red) obtained from lysates of HeLa cells transfected with pYX017 and the indicated siRNA; technical replicates are side-by-side; biological replicates are indicated with subscripts
Mentions: While investigating the effect of p38δ on L1 retrotransposition, we performed siRNA experiments using a SMARTpool mixture against p38δ (Dharmacon, M-003591-02-0005) and the NTC siRNA #3 (Dharmacon). Although the siRNA against p38δ dramatically reduced the number of G418-resistant colonies relative to NTC #3, RT-PCR showed no significant knockdown of the p38δ transcript (data not shown). Interestingly, however, NTC #3 considerably increased colony density relative to the mock control (Fig. 6a left). EGFP fluorescence from cells pretreated with siRNA prior to transfection suggested that the siRNA had little impact on transfection efficiency (Fig. 6a right). Given these unexpected results, we tested an additional control siRNA, NTC #5, also from Dharmacon. In marked contrast to NTC #3, NTC #5 dramatically reduced G418-resistant colonies relative to the mock control (Fig. 6b top). Neither NTC dramatically affected cell growth, though NTC #3 had a slight inhibitory effect (Fig. 6b bottom). It is notable that unlike p38δ-WT, the NTC siRNAs exerted their respective effects similarly on both Fluc luminescence and G418-resistant colony formation (Fig. 6b top, c left and d). However, L1 activity as reported by the Fluc/Rluc ratio appears to be decreased by NTC #3 rather than increased (Fig. 6c). We did not further investigate potential causes for these results. Information on Dharmacon’s website states that each NTC is reported to contain a minimum of 4 mismatches to all human, mouse and rat genes and to have minimal effects in genome-wide targeting via microarray analyses. We did not test Dharmacon’s NTC #1, as it was reported to increase cell growth (personal communication, Dharmacon), nor NTC #2 or #4 due to their targeting of Firefly luciferase (Dharmacon website).Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.

Results: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.

Conclusions: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

No MeSH data available.


Related in: MedlinePlus