Organ-, sex- and age-dependent patterns of endogenous L1 mRNA expression at a single locus resolution.

Expression of L1 mRNA, the first step in the L1 copy-and-paste amplification cycle, is a prerequisite for L1-associated genomic instability. We used a reported stringent bioinformatics method to parse L1 mRNA transcripts and measure the level of L1 mRNA expressed in mouse and rat organs at a locus-specific resolution. This analysis determined that mRNA expression of L1 loci in rodents exhibits striking organ specificity with less than 0.8% of loci shared between organs of the same organism. This organ specificity in L1 mRNA expression is preserved in male and female mice and across age groups. We discovered notable differences in L1 mRNA expression between sexes with only 5% of expressed L1 loci shared between male and female mice. Moreover, we report that the levels of total L1 mRNA expression and the number and spectrum of expressed L1 loci fluctuate with age as independent variables, demonstrating different patterns in different organs and sexes. Overall, our comparisons between organs and sexes and across ages ranging from 2 to 22 months establish previously unforeseen dynamic changes in L1 mRNA expression in vivo. These findings establish the beginning of an atlas of endogenous L1 mRNA expression across a broad range of biological variables that will guide future studies.

A comprehensive approach to expression of L1 loci.

L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the 'authentic' L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues.

Potential for genomic instability associated with retrotranspositionally-incompetent L1 loci.

Expression of the L1 retrotransposon can damage the genome through insertional mutagenesis and the generation of DNA double-strand breaks (DSBs). The majority of L1 loci in the human genome are 5'-truncated and therefore incapable of retrotransposition. While thousands of full-length L1 loci remain, most are retrotranspositionally-incompetent due to inactivating mutations. However, mutations leading to premature stop codons within the L1 ORF2 sequence may yield truncated proteins that retain a functional endonuclease domain. We demonstrate that some truncated ORF2 proteins cause varying levels of toxicity and DNA damage when chronically overexpressed in mammalian cells. Furthermore, transfection of some ORF2 constructs containing premature stop codons supported low levels of Alu retrotransposition, demonstrating the potential for select retrotranspositionally-incompetent L1 loci to generate genomic instability. This result suggests yet another plausible explanation for the relative success of Alu elements in populating the human genome. Our data suggest that a subset of retrotranspositionally-incompetent L1s, previously considered to be harmless to genomic integrity, may have the potential to cause chronic DNA damage by introducing DSBs and mobilizing Alu. These results imply that the number of known L1 loci in the human genome that potentially threaten its stability may not be limited to the retrotranspositionally active loci.

Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities.

L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.

The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor.

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.

The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations.

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.

Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein.

BACKGROUND: LINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Ongoing L1 retrotransposition in the germ line and somatic tissues has contributed to structural genomic variations and disease-causing mutations in the human genome. L1 mobilization relies on the function of two, self-encoded proteins, ORF1 and ORF2. The ORF2 protein contains two characterized domains: endonuclease and reverse transcriptase. RESULTS: Using a bacterially purified endonuclease domain of the human L1 ORF2 protein, we have generated a monoclonal antibody specific to the human ORF2 protein. We determined that the epitope recognized by this monoclonal antibody includes amino acid 205, which is required for the function of the L1 ORF2 protein endonuclease. Using an in vitro L1 cleavage assay, we demonstrate that the monoclonal anti-ORF2 protein antibody partially inhibits L1 endonuclease activity without having any effect on the in vitro activity of the human AP endonuclease. CONCLUSIONS: Overall, our data demonstrate that this anti-ORF2 protein monoclonal antibody is a useful tool for human L1-related studies and that it provides a rationale for the development of antibody-based inhibitors of L1-induced damage.