Lixin Dai

Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.

LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotranspositions mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

Long Interspersed Element-1 Protein Expression Is a Hallmark of Many Human Cancers

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition.

BackgroundLINE-1s (L1, Long Interspersed Element-1) are the most abundant autonomous non-LTR retrotransposons in the human genome and replicate by reverse transcription of an RNA intermediate. Full-length L1 encodes two open reading frames (ORF1, ORF2) and ORF2 has reverse transcriptase activity.ResultsHere we expressed human L1 RT in E. coli and the purified protein displayed the same RT activity as that of ORF2p expressed in insect cells. We tested the effect of different reverse transcriptase inhibitors on L1 RT and found that all four tested nucleoside inhibitors efficiently inhibited L1 RT activity competitively. The Ki values of NRTIs were calculated (AZTTP, 16.4 ± 4.21 nM; d4TTP, 0.73 ± 0.22 nM; ddCTP, 0.72 ± 0.16 nM; 3TCTP, 12.9 ± 2.07 nM). L1 RT was less sensitive to non-nucleoside reverse transcriptase inhibitors, among these nevirapine had no effect, even at concentrations up to 500 μM. We also examined the effect of RT inhibitors on L1 retrotransposition efficiency in vivo using a cell-based retrotransposition assay. Similarly, all analog inhibitors decreased L1 retrotransposition frequency with different potencies whereas nevirapine had little or no effect on L1 retrotransposition. For comparison, we also tested the same inhibitors to highly purified RT of an LTR-retrotransposon (Ty1) and found it was less sensitive to NRTIs than L1 RT and has the same inhibition profile as L1 RT to NNRTIs.ConclusionsThese data indicate that bacterially expressed L1 RT is an active reverse transcriptase sensitive to nucleoside RT inhibitors but not to non-nucleoside inhibitors.

Architectural Organization of the Metabolic Regulatory Enzyme Ghrelin O-Acyltransferase

Background: Ghrelin O-acyltransferase (GOAT) is a membrane protein that is responsible for octanoylating the metabolism-regulating peptide hormone ghrelin. Results: We have used a combination of approaches to determine the topology of GOAT. Conclusion: We have shown that GOAT has 11 transmembrane-spanning domains and one reentrant loop. Significance: These findings serve as a reference for other membrane-bound O-acyltransferase family members. Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes.

Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation.

Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation

ABSTRACT Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell.

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis.

Expression and detection of LINE-1 ORF-encoded proteins

LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

TE-array—a high throughput tool to study transposon transcription

BackgroundAlthough transposable element (TE) derived DNA accounts for more than half of mammalian genomes and initiates a significant proportion of RNA transcripts, high throughput methods are rarely leveraged specifically to detect expression from interspersed repeats.ResultsTo characterize the contribution of transposons to mammalian transcriptomes, we developed a custom microarray platform with probes covering known human and mouse transposons in both sense and antisense orientations. We termed this platform the “TE-array” and profiled TE repeat expression in a panel of normal mouse tissues. Validation with nanoString® and RNAseq technologies demonstrated that TE-array is an effective method. Our data show that TE transcription occurs preferentially from the sense strand and is regulated in highly tissue-specific patterns.ConclusionsOur results are consistent with the hypothesis that transposon RNAs frequently originate within genomic TE units and do not primarily accumulate as a consequence of random ‘read-through’ from gene promoters. Moreover, we find TE expression is highly dependent on the tissue context. This suggests that TE expression may be related to tissue-specific chromatin states or cellular phenotypes. We anticipate that TE-array will provide a scalable method to characterize transposable element RNAs.

Characterization of L1-Ribonucleoprotein Particles.

The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR.