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The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding.

ABSTRACT: The human LINE-1/L1 ORF2 protein is a multifunctional enzyme which plays a vital role in the life cycle of the human L1 retrotransposon. The protein consists of an endonuclease domain, followed by a central reverse transcriptase domain and a carboxy-terminal C-domain with unknown function. Here, we explore the nucleic acid binding properties of the 180-amino acid carboxy-terminal segment (CTS) of the human L1 ORF2p in vitro. In a series of experiments involving gel shift assay, we demonstrate that the CTS of L1 ORF2p binds RNA in non-sequence-specific manner. Finally, we report that mutations destroying the putative Zn-knuckle structure of the protein do not significantly affect the level of RNA binding and discuss the possible functional role of the CTS in L1 retrotransposition.

SUBMITTER: Piskareva O

PROVIDER: S-EPMC3821027 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding.

Piskareva Olga O Ernst Christina C Higgins Niamh N Schmatchenko Vadim V

FEBS open bio 20130921

The human LINE-1/L1 ORF2 protein is a multifunctional enzyme which plays a vital role in the life cycle of the human L1 retrotransposon. The protein consists of an endonuclease domain, followed by a central reverse transcriptase domain and a carboxy-terminal C-domain with unknown function. Here, we explore the nucleic acid binding properties of the 180-amino acid carboxy-terminal segment (CTS) of the human L1 ORF2p in vitro. In a series of experiments involving gel shift assay, we demonstrate th ...[more]

PMID: 24251107

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Project description:BACKGROUND:Approximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription. RESULTS:Bioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity. CONCLUSIONS:Overall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.

Dataset Information

The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding.

Publications

The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding.

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