The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2. Tuberous sclerosis complex (TSC)5 is an autosomal dominant genetic disorder affecting 1 in 6,000 -10,000 births (1). Mutations in either of the two genes, TSC1 (also called hamartin) or TSC2 (tuberin), cause the disorder characterized by benign tumor formation (hamartomas) in various organs and tissues. Complications of hamartomas in critical organs include renal failure, seizures, mental retardation, and autism (1). One of the hallmarks of TSC hamartomas is an increase in cell size, implicating TSC1 and TSC2 as negative regulators of cell growth (2-4).Recent studies have revealed the molecular mechanism for the tumor suppressor function of TSC1 and TSC2, which form a physical and functional complex (5). The TSC1⅐TSC2 complex suppresses cell growth by inhibiting the mammalian target of rapamycin, mTOR, which is a central controller of cell growth. TSC1/ TSC2 has GTPase-activating protein (GAP) activity toward the Rheb small GTPase (6, 7). Rheb acts upstream of and stimulates mTOR. TSC2 is the catalytic GAP subunit, while TSC1 enhances TSC2 function by stabilizing TSC2. The majority of disease-associated TSC1 mutations identified result in no TSC1 protein being expressed; therefore, the free TSC2 protein in TSC1 mutant cells is unstable (1). Similarly, many disease-derived TSC2 mutants are unstable due to weakened interaction with TSC1 (8, 9). However, the precise mechanism how TSC1 stabilizes TSC2 is largely unclear.In this report, we identified HERC1 as a TSC2-interacting protein. The COOH-terminal region of HERC1 has a HECT E3 ubiquitin ligase domain (10). Interestingly, HERC1 does not associate with TSC1. Moreover, TSC1 efficiently competes with HERC1 for TSC2 binding. Our study provides a potential biochemical mechanism of TSC1 in TSC2 stabilization by inhibiting the interaction between TSC2 and the E3 ubiquitin ligase HERC1. EXPERIMENTAL PROCEDURESPlasmids and Antibodies-HA-TSC2 and Myc-TSC1 we...

Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80–100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.

Transposable elements (TEs) or "jumping genes" historically have been disparaged as a class of "junk DNA" in mammalian genomes (1,2). The advent of whole genome DNA sequencing, in conjunction with molecular genetic, biochemical, and modern genomic and functional studies, is revealing that TEs are biologically important components of mammalian genomes. TEs are classified by whether they mobilize via a DNA or an RNA intermediate (detailed in (3)). Classical DNA transposons, such as the maize Activator/ Dissociation (Ac/Ds) elements originally discovered by Barbara McClintock, move via a DNA intermediate (4,5). Their mobility (i.e., transposition) can impact organismal phenotypes such as corn kernel variegation. Retrotransposons, the predominant class of TEs in most mammalian genomes, mobilize via an RNA intermediate by a process termed retrotransposition (6).The completion of the human genome reference sequence (HGR) (7, 8) confirmed the results of DNA hybridization-based re-annealing studies (9, 10) and revealed that retrotransposons have been a major force in shaping the structure and function of mammalian genomes. The mobility of non-long terminal repeat (non-LTR) retrotransposons, namely autonomously active Long INterspersed Element-1 sequences (LINE-1s, also known proteins can work efficiently in trans, it is formally possible that trans-complementation might allow the assembly of functional virus-like particles from partially defective HERVs, allowing the generation of new retrotransposition events. Advances in DNA sequencing technologies may reveal rare, active HERV-K elements or de novo germline or somatic HERV-K retrotransposition events in individual human genomes.LTR-retrotransposons are present at greater than 600,000 copies in mouse DNA and comprise approximately 10% of the genome (21). In contrast to the human genome, the mouse genome contains multiple, active ERV subfamilies (reviewed in (50, 51)). These include autonomously active Mus D and intracisternal A particle (IAP) elements, as well as non-autonomous early transposons (ETns) and mammalian apparent LTR retrotransposons (MALRs). It is estimated that ERV insertions are responsible for approximately ten percent of spontaneously arising mouse mutations (reviewed in (51)) (discussed in greater detail in other Chapters of Mobile DNA III). LINE-1 Retrotransposons: abundance and structureA brief overview of human LINE-1 evolution and nomenclature-LINE-1 retrotransposons have been amplifying in mammalian genomes for greater than 160 million years (52-54). In humans, the vast majority of LINE-1 sequences have amplified since the divergence of the ancestral mouse and human lineages approximately 65-75 million years ago (7). As a consequence, LINE-1-derived sequences now account for approximately 17% of human genomic DNA (7) (Figure 1).Sequence comparisons between individual genomic LINE-1 sequences and a consensus sequence derived from modern, active LINE-1s can be used to estimate the age of genomic LINE-1s. These analyses uncovered sixteen LINE-1 pri...