LINE-1 Retrotransposition Requires the Nucleic Acid Chaperone Activity of the ORF1 Protein

Martin, Sandra L.; Cruceanu, Margareta; Branciforte, Dan; Li, Patrick Wai-lun; Kwok, Stanley C.; Hodges, Robert S.; Williams, Mark C.

doi:10.1016/j.jmb.2005.03.003

Cited by 158 publications

(206 citation statements)

References 31 publications

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“…The observation that RNA interference against L1-ORF1p induces morphologic differentiation, reduces proliferation, and decreases the expression of some genes (8,9) suggests that L1-ORF1p might have features of a transcription factor. ORF1p has been reported to be an RNA binding protein (10), and more recent evidence is in accordance with the hypothesis that L1-ORF1p also binds ssDNA (11,12).…”

supporting

confidence: 81%

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Neidhart

Karouzakis

Schumann

2010

Arthritis & Rheumatism

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Objective. To explore whether the increased expression of long interspersed nuclear element 1 (LINE-1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex-1, an exonuclease involved in the metabolization of L1 DNA: RNA hybrids.Methods. Chromatin immunoprecipitation was used to detect L1-related p40 protein (L1-ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1-ORF1p and pGL3-TS3 carrying the target sequence for L1-ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex-1 measured by flow cytometry. The expression of Trex-1 mRNA and protein was also compared using real-time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex-1 in the L1-ORF1p-mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex-1 expression vector.Results. Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3-TS3. L1-ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex-1. Levels of Trex-1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex-1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex-1, however, resulted in an enhancement of L1-ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex-1 inhibited 5-azacytidine-induced expression of p38␦ MAPK, a gene carrying the TS3 sequence. Conclusion. The deficiency of Trex-1 in RASFs allows a longer half-life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a "spontaneous" aggressive behavior.Trex-1 is the major 3Ј35Ј DNA exonuclease in mammalian cells, and mutations in the human trex-1 gene can cause Aicardi-Goutières syndrome (1), a disease that is characterized by perturbed immunity. Such mutations also appear to be involved in systemic lupus erythematosus (SLE) (2), in which the failure to process intermediates of nucleic acid metabolism can result in the activation of uncontrolled autoimmunity. Trex-1 prevents the accumulation of single-stranded DNA (ssDNA) fragments derived from endogenous long interspersed nuclear element 1 (LINE-1; L1) retrotransposons (3), the expression of which has been reported in rheumatoid arthritis synovial fibroblasts (RASFs) (4). The promoter of the L1 retroelement is hypomethylated in RASFs (5), reflecting global DNA hypomethylation. The L1-related p40 protein (L1-ORF1p) has nucleic acid binding properties and favors the transcription of specific genes (6). The L1-encoded p150 protein (L1-ORF2p) has a reverse transcription acti...

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supporting

confidence: 81%

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Neidhart

Karouzakis

Schumann

2010

Arthritis & Rheumatism

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“…Many of the exchanged arginine residues may solely make contacts to the phosphate-ribose backbone of the RNA, thereby fixing its conformation. R159 and R261, however, appear particularly important, as they are invariant in sequence alignments and are functionally required at several steps in retrotransposition (14,16,17,45). They may be involved in multiple contacts, possibly stacking on bases or locking the relative orientations of the RRM and CTD domains on the RNA.…”

Section: The Crystal Structure Of the Rrm Domain In Human L1orf1p Showsmentioning

confidence: 99%

Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame

Khazina

Weichenrieder

2009

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

137

200

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Non-LTR retrotransposons (NLRs) are a unique class of mobile genetic elements that have significant impact on the evolution of eukaryotic genomes. However, the molecular details and functions of their encoded proteins, in particular of the accessory ORF1p proteins, are poorly understood. Here, we identify noncanonical RNA-recognitionmotifs (RRMs) in several phylogenetically unrelated NLR ORF1p proteins. This provides an explanation for their RNA-binding properties and clearly shows that they are not related to the retroviral nucleocapsid protein Gag, despite the frequent presence of CCHC zinc knuckles. In particular, we characterize the ORF1p protein of the human long interspersed nuclear element 1 (LINE-1 or L1). We show that L1ORF1p is a multidomain protein, consisting of a coiled coil (cc), RRM, and C-terminal domain (CTD). Most importantly, we solved the crystal structure of the RRM domain, which is characterized by extended loops stabilized by unique salt bridges. Furthermore, we demonstrate that L1ORF1p trimerizes via its N-terminal cc domain, and we suggest that this property is functionally important for all homologues. The formation of distinct complexes with singlestranded nucleic acids requires the presence of the RRM and CTD domains on the same polypeptide chain as well as their close cooperation. Finally, the phylogenetic analysis of mammalian L1ORF1p shows an ancient origin of the RRM domain and supports a modular evolution of NLRs.genome evolution ͉ LINE-1 ͉ nucleic acid chaperone ͉ RNP ͉ crystal structure

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“…The increase in the transition width and the extent of hysteresis are the features that distinguish the nucleic acid chaperone proteins studied in our laboratory from other nucleic acid binding proteins (ref. (9,31,32) and unpublished results).…”

Section: Introductionmentioning

confidence: 62%

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

Cruceanu

Stephen

Beuning

et al. 2006

Analytical Biochemistry

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We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.

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LINE-1 Retrotransposition Requires the Nucleic Acid Chaperone Activity of the ORF1 Protein

Cited by 158 publications

References 31 publications

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

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