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

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-recognition-motifs (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 single-stranded 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.

[1]  Sandra L. Martin,et al.  The ORF1 Protein Encoded by LINE-1: Structure and Function During L1 Retrotransposition , 2006, Journal of biomedicine & biotechnology.

[2]  H. Kazazian,et al.  Retrotransposons Revisited: The Restraint and Rehabilitation of Parasites , 2008, Cell.

[3]  S. Boissinot,et al.  L1 (LINE-1) retrotransposon diversity differs dramatically between mammals and fish. , 2004, Trends in genetics : TIG.

[4]  H. Kazazian,et al.  LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex , 2007, Molecular and Cellular Biology.

[5]  Jef D. Boeke,et al.  Human L1 Retrotransposition: cisPreference versus trans Complementation , 2001, Molecular and Cellular Biology.

[6]  D. Finnegan,et al.  A LINE‐like transposable element in Drosophila, the I factor, encodes a protein with properties similar to those of retroviral nucleocapsids , 1997, The EMBO journal.

[7]  R. Löwer,et al.  Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells , 2007, Nucleic acids research.

[8]  Michael Müller,et al.  Thermodynamic characterization of an engineered tetracycline-binding riboswitch , 2006, Nucleic acids research.

[9]  Johannes Söding,et al.  The HHpred interactive server for protein homology detection and structure prediction , 2005, Nucleic Acids Res..

[10]  Jef D Boeke,et al.  Human L1 element target‐primed reverse transcription in vitro , 2002, The EMBO journal.

[11]  Jeffrey S. Han,et al.  LINE‐1 retrotransposons: Modulators of quantity and quality of mammalian gene expression? , 2005, BioEssays : news and reviews in molecular, cellular and developmental biology.

[12]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[13]  N. Okada,et al.  Isolation and characterization of retrotransposition-competent LINEs from zebrafish. , 2006, Gene.

[14]  Gabriele Varani,et al.  RNA is rarely at a loss for companions; as soon as RNA , 2008 .

[15]  D. Carroll,et al.  Composite transposable elements in the Xenopus laevis genome. , 1989, Molecular and cellular biology.

[16]  J. V. Moran,et al.  Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition. , 2005, Human molecular genetics.

[17]  O. Weichenrieder,et al.  APE-type non-LTR retrotransposons: determinants involved in target site recognition , 2005, Cytogenetic and Genome Research.

[18]  T. Eickbush,et al.  Origins and Evolution of Retrotransposons , 2002 .

[19]  F. Bushman,et al.  Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon , 2001, Molecular and Cellular Biology.

[20]  Jef D Boeke,et al.  High Frequency Retrotransposition in Cultured Mammalian Cells , 1996, Cell.

[21]  G. Hannon,et al.  The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race , 2007, Science.

[22]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[23]  Eric Blanc,et al.  Automated structure solution with autoSHARP. , 2007, Methods in molecular biology.

[24]  Jef D Boeke,et al.  Human L1 Retrotransposon Encodes a Conserved Endonuclease Required for Retrotransposition , 1996, Cell.

[25]  S. Martin,et al.  Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells , 1991, Molecular and cellular biology.

[26]  S. Martin,et al.  Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1. , 2000, Journal of molecular biology.

[27]  J. Feigon,et al.  Identification and Solution Structure of a Highly Conserved C-terminal Domain within ORF1p Required for Retrotransposition of Long Interspersed Nuclear Element-1* , 2007, Journal of Biological Chemistry.

[28]  International Human Genome Sequencing Consortium Initial sequencing and analysis of the human genome , 2001, Nature.

[29]  D. Keller,et al.  Trimeric structure for an essential protein in L1 retrotransposition , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. V. Moran,et al.  Mammalian LINE-1 Retrotransposons and Related Elements , 2002 .

[31]  Oliver Weichenrieder,et al.  Structure and assembly of the Alu domain of the mammalian signal recognition particle , 2000, Nature.

[32]  T. Eickbush,et al.  Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: A mechanism for non-LTR retrotransposition , 1993, Cell.

[33]  Y. Shamoo,et al.  Multiple RNA binding domains (RBDs) just don't add up. , 1995, Nucleic acids research.

[34]  C. Brun,et al.  In vivo RNA localization of I factor, a non-LTR retrotransposon, requires a cis-acting signal in ORF2 and ORF1 protein , 2005, Nucleic acids research.

[35]  C. Lister,et al.  Transposable elements controlling I-R hybrid dysgenesis in D. melanogaster are similar to mammalian LINEs , 1986, Cell.

[36]  H. Hohjoh,et al.  Sequence‐specific single‐strand RNA binding protein encoded by the human LINE‐1 retrotransposon , 1997, The EMBO journal.

[37]  S. Martin,et al.  In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. V. Moran,et al.  Exon shuffling by L1 retrotransposition. , 1999, Science.

[39]  M. Pardue,et al.  Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  C. Dominguez,et al.  The RNA recognition motif, a plastic RNA‐binding platform to regulate post‐transcriptional gene expression , 2005, The FEBS journal.

[41]  Pavlos Progias,et al.  A conserved trimerization motif controls the topology of short coiled coils. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[42]  O. Weichenrieder,et al.  Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon. , 2004, Structure.

[43]  H. Hohjoh,et al.  Cytoplasmic ribonucleoprotein complexes containing human LINE‐1 protein and RNA. , 1996, The EMBO journal.

[44]  Richard J Morris,et al.  Towards complete validated models in the next generation of ARP/wARP. , 2004, Acta crystallographica. Section D, Biological crystallography.

[45]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[46]  D. Keller,et al.  Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition. , 2006, Journal of molecular biology.

[47]  K. Kojima,et al.  Cross-genome screening of novel sequence-specific non-LTR retrotransposons: various multicopy RNA genes and microsatellites are selected as targets. , 2003, Molecular biology and evolution.

[48]  R. Hodges,et al.  LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. , 2005, Journal of molecular biology.

[49]  J. Jurka,et al.  The Esterase and PHD Domains in CR1-Like Non-LTR Retrotransposons , 2003, Molecular biology and evolution.

[50]  J. Jurka,et al.  Repbase Update, a database of eukaryotic repetitive elements , 2005, Cytogenetic and Genome Research.

[51]  A. Athanasiadis,et al.  Intracellular Targeting of Gag Proteins of the Drosophila Telomeric Retrotransposons , 2003, Journal of Virology.

[52]  D. Voytas,et al.  Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana. , 1996, Genetics.

[53]  J. Biedler,et al.  Non-LTR retrotransposons in the African malaria mosquito, Anopheles gambiae: unprecedented diversity and evidence of recent activity. , 2003, Molecular biology and evolution.