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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.


The MAPK p38δ phosphorylates ORF1p on S/T-P motifs required for L1 retrotransposition. a ORF1p-WT or S/T-P mutants (200 μM), purified from E. coli, were incubated with 85 nM activated p38δ-WT (top) or the constitutively active p38δ mutant F324S (bottom) in the presence of [γ-32P]-ATP; bands on autoradiogram show 32P incorporation into ORF1p. ORF1p mutants are S18A/S27A/T203G/T213G (AAGG), S18A/S27A (AA), T203G/T213G (GG), S27A/T203G/T213G (SAGG), S18A/T203G/T213G (ASGG), S18A/S27A/T213G (AATG) and S18A/S27A/T203G (AAGT). b ORF1p-WT was incubated with activated p38δ-WT, p38δ-F324S, an inactive p38δ mutant D176A, or no kinase in reactions as described in (a). c A Coomassie-stained gel shows each ORF1p construct (approximately 100 ng) purified from E. coli.
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Fig1: The MAPK p38δ phosphorylates ORF1p on S/T-P motifs required for L1 retrotransposition. a ORF1p-WT or S/T-P mutants (200 μM), purified from E. coli, were incubated with 85 nM activated p38δ-WT (top) or the constitutively active p38δ mutant F324S (bottom) in the presence of [γ-32P]-ATP; bands on autoradiogram show 32P incorporation into ORF1p. ORF1p mutants are S18A/S27A/T203G/T213G (AAGG), S18A/S27A (AA), T203G/T213G (GG), S27A/T203G/T213G (SAGG), S18A/T203G/T213G (ASGG), S18A/S27A/T213G (AATG) and S18A/S27A/T203G (AAGT). b ORF1p-WT was incubated with activated p38δ-WT, p38δ-F324S, an inactive p38δ mutant D176A, or no kinase in reactions as described in (a). c A Coomassie-stained gel shows each ORF1p construct (approximately 100 ng) purified from E. coli.

Mentions: We first determined whether activated wild type p38δ (WT, Invitrogen) could phosphorylate ORF1p on its S/T-P motifs, which are required for robust L1 activity [31]. In vitro radioactive kinase assays revealed that p38δ-WT exclusively phosphorylated bacterially purified ORF1p on these residues, as an ORF1p carrying mutations at all four motifs, S18A/S27A/T203G/T213G (AAGG), was not phosphorylated (Fig. 1a top). We next tested the ability of p38δ-WT to phosphorylate the ORF1p mutants S18A/S27A (AA) and T203G/T213G (GG), and found that the majority of phosphorylation occurred on the GG mutant, which retained both serine motifs (Fig. 1a top).Fig. 1


Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
The MAPK p38δ phosphorylates ORF1p on S/T-P motifs required for L1 retrotransposition. a ORF1p-WT or S/T-P mutants (200 μM), purified from E. coli, were incubated with 85 nM activated p38δ-WT (top) or the constitutively active p38δ mutant F324S (bottom) in the presence of [γ-32P]-ATP; bands on autoradiogram show 32P incorporation into ORF1p. ORF1p mutants are S18A/S27A/T203G/T213G (AAGG), S18A/S27A (AA), T203G/T213G (GG), S27A/T203G/T213G (SAGG), S18A/T203G/T213G (ASGG), S18A/S27A/T213G (AATG) and S18A/S27A/T203G (AAGT). b ORF1p-WT was incubated with activated p38δ-WT, p38δ-F324S, an inactive p38δ mutant D176A, or no kinase in reactions as described in (a). c A Coomassie-stained gel shows each ORF1p construct (approximately 100 ng) purified from E. coli.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120415&req=5

Fig1: The MAPK p38δ phosphorylates ORF1p on S/T-P motifs required for L1 retrotransposition. a ORF1p-WT or S/T-P mutants (200 μM), purified from E. coli, were incubated with 85 nM activated p38δ-WT (top) or the constitutively active p38δ mutant F324S (bottom) in the presence of [γ-32P]-ATP; bands on autoradiogram show 32P incorporation into ORF1p. ORF1p mutants are S18A/S27A/T203G/T213G (AAGG), S18A/S27A (AA), T203G/T213G (GG), S27A/T203G/T213G (SAGG), S18A/T203G/T213G (ASGG), S18A/S27A/T213G (AATG) and S18A/S27A/T203G (AAGT). b ORF1p-WT was incubated with activated p38δ-WT, p38δ-F324S, an inactive p38δ mutant D176A, or no kinase in reactions as described in (a). c A Coomassie-stained gel shows each ORF1p construct (approximately 100 ng) purified from E. coli.
Mentions: We first determined whether activated wild type p38δ (WT, Invitrogen) could phosphorylate ORF1p on its S/T-P motifs, which are required for robust L1 activity [31]. In vitro radioactive kinase assays revealed that p38δ-WT exclusively phosphorylated bacterially purified ORF1p on these residues, as an ORF1p carrying mutations at all four motifs, S18A/S27A/T203G/T213G (AAGG), was not phosphorylated (Fig. 1a top). We next tested the ability of p38δ-WT to phosphorylate the ORF1p mutants S18A/S27A (AA) and T203G/T213G (GG), and found that the majority of phosphorylation occurred on the GG mutant, which retained both serine motifs (Fig. 1a top).Fig. 1

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.