Cellular RNA Molecules Research Articles

RNA structure promotes liquid-to-solid phase transition of short RNAs in neuronal dysfunction

In nucleotide expansion disorders, RNA foci are reportedly associated with neurodegenerative disease pathogeneses. Characteristically, these RNAs exhibit long poly-RNA repeats, such as 47 × CAG, 47 × CUG, or 29 × GGGGCC, usually becoming abnormal pathological aggregations above a critical number of nucleotide repeats. However, it remains unclear whether short, predominantly cellular RNA molecules can cause phase transitions to induce RNA foci. Herein, we demonstrated that short RNAs even with only two repeats can aggregate into a solid-like state via special RNA G-quadruplex structures. In human cells, these solid RNA foci could not dissolve even when using agents that disrupt RNA gelation. The aggregation of shorter RNAs can be clearly observed in vivo. Furthermore, we found that RNA foci induce colocalization of the RNA-binding protein Sam68, a protein commonly found in patients with fragile X-associated tremor/ataxia syndrome, suppressing cell clonogenicity and eventually causing cell death. Our results suggest that short RNA gelation promoted by specific RNA structures contribute to the neurological diseases, which disturb functional cellular processes.

Optimizing Conditions for the Production of Bacterial Extracellular Vesicles of Vibrio vulnificus and Analysis of the Inner Small RNA Compositions.

Chemical and physical elements affecting the production of bacterial extracellular vesicles (BEVs) of the human pathogen Vibrio vulnificus were quantitatively assessed to optimize the conditions for the BEV production by using the western blot quantification for an outer membrane porin OmpU and by fluorescent dye FM4-64. When cells were cultured at 37°C in an enriched medium (2 × Luria Bertani; 2 × LB) in the presence of EDTA, they produced about 70% more BEVs. BEVs were purified from the cells cultured in the established optimal conditions by the density gradient ultracentrifugation. The dynamic light scattering measurement of the purified BEVs showed that the diameter of them ranged from approximately 25 nm to 161 nm. We hypothesized that there may be some features in nucleotide sequences specific to RNAs packaged in BEVs compared to those in cellular RNA molecules. We compared the nucleotide sequences and abundance of sRNAs between in the cellular fraction and in BEVs through next-generation sequencing (NGS). While no distinct feature was observed in the nucleotide sequences of sRNAs between two groups, the length of sRNA fragments from BEVs were significantly shorter than those in cytoplasm.

Theory of HIV-1 RNA genome selection.

It is not understood how the HIV-1 retrovirus selects it genomic RNA (gRNA) for packaging into virions from among the multitude of cellular RNA molecules in the cytoplasm of infected cells. Under in-vitro conditions, the Gag proteins of HIV-1 have only a very weak specific binding affinity for the RNA Ψ sequence, which has been shown to be the key recognition label of genomic RNA. In addition, the cellular gRNA concentration is very low yet HIV packages gRNA with high fidelity. We present a numerical model for kinetic HIV-1 gRNA selection that is based on fusion of precursor buds followed by gRNA dimerization.

Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

Hepatic RNA adduction derived from metabolic activation of retrorsine in vitro and in vivo

Pyrrolizidine alkaloids (PAs) are among the most significant hepatotoxins widely distributed in plant species. Incidence of liver injuries caused by PAs has been reported worldwide, and the reactive metabolites of PAs are known to play a critical role in causing the hepatotoxicity. To better understand the toxicity-induction mechanisms, we explored the interactions of PA metabolites with cellular RNA molecules, and examined their effects on the biochemical and metabolic properties of hepatic RNAs. After exposure to retrorsine, adduction on adenosine and guanosine were detected in mouse liver microsomal incubations, cultured mouse primary hepatocytes, and mouse liver tissues. NMR analysis showed that the exocyclic amino group participated in the adduction. We found drastically altered properties and metabolism of the adducted RNA such as reverse-transcriptability, translatability, and RNase-susceptibility. In addition, endogenous modification of N6-methyladenosine (m6A) was remarkably reduced.

The occurrence order and cross-talk of different tRNA modifications.

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides, which greatly increase the complexity and utility of RNAs. Transfer RNAs (tRNAs) are the most heavily modified cellular RNA molecules and contain the largest variety of modifications. Modification of tRNAs is pivotal for protein synthesis and also precisely regulates the noncanonical functions of tRNAs. Defects in tRNA modifications lead to numerous human diseases. Up to now, more than 100 types of modifications have been found in tRNAs. Intriguingly, some modifications occur widely on all tRNAs, while others only occur on a subgroup of tRNAs or even only a specific tRNA. The modification frequency of each tRNA is approximately 7% to 25%, with 5-20 modification sites present on each tRNA. The occurrence and modulation of tRNA modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered. In particular, tRNA modifications are responsive to environmental changes, indicating their dynamic and highly organized nature. In this review, we summarized the known occurrence order, cross-talk, and cooperativity of tRNA modifications.

Generation of 2′,3′-Cyclic Phosphate-Containing RNAs as a Hidden Layer of the Transcriptome

Cellular RNA molecules contain phosphate or hydroxyl ends. A 2′,3′-cyclic phosphate (cP) is one of the 3′-terminal forms of RNAs mainly generated from RNA cleavage by ribonucleases. Although transcriptome profiling using RNA-seq has become a ubiquitous tool in biological and medical research, cP-containing RNAs (cP-RNAs) form a hidden transcriptome layer, which is infrequently recognized and characterized, because standard RNA-seq is unable to capture them. Despite cP-RNAs’ invisibility in RNA-seq data, increasing evidence indicates that they are not accumulated simply as non-functional degradation products; rather, they have physiological roles in various biological processes, designating them as noteworthy functional molecules. This review summarizes our current knowledge of cP-RNA biogenesis pathways and their catalytic enzymatic activities, discusses how the cP-RNA generation affects biological processes, and explores future directions to further investigate cP-RNA biology.

Selective Packaging of HIV-1 RNA by GAG Proteins

It is not understood how the HIV-1 retrovirus selects it genomic RNA (gRNA) for packaging into virions from among the multitude of cellular RNA molecules in the cytoplasm of infected cells. Under in vitro conditions, the Gag proteins of HIV-1 have no specific binding affinity for the RNA Ψ sequence, which has been shown to be the key recognition element of genomic RNA. We propose a search mechanism based on the two-state nature of the Gag proteins in solution. The minimum free energy state is the B (or “bent”) state. In this state, Gag is non-specifically bound to RNA molecules through the NC and MA cationic subdomains and mobile. Next, in the E (or “extended”) state, only the NC domain of Gag binds to the RNA molecule that is being searched, while the MA domain may bind to distant regions of RNA, small RNAs, small molecules such as PIP, or the plasma membrane. In the E state, Gag is immobile and has an enhanced binding affinity for the Ψ sequence (1,2). In addition, unlike the B-state form E-state Gag has a binding interface for other E-state Gags, thereby allowing stabilization of E state by multimerization and eventual capsid assembly. Alternation between B and E state Gag allows for an efficient search mechanism that is similar to that of transcription factor proteins searching DNA (3). (3) Slutsky, et.al. 2004. Kinetics of protein-DNA interaction: facilitated target location in sequence-dependent potential. Biophysical journal, 87(6), pp.4021-4035. (1)Webb, et.al. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging. RNA. 2013 (8):pp.1078-88. (2) Comas-Garcia, et.al. 2017. Dissection of specific binding of HIV-1 Gag to the “packaging signal” in viral RNA. eLife, 6, p.27055.

Nonradioactive Detection of Small RNAs Using Digoxigenin-Labeled Probes.

Small RNAs have been traditionally detected and quantified using small RNA blots, a modified Northern blot technique. The small RNAs are size-fractionated from the rest of the cellular RNA molecules by polyacrylamide gel electrophoresis and transferred by blotting onto a positively charged membrane. A radiolabeled probe was then traditionally used to detect a specific small RNA in the cellular pool. Small RNA blotting is a relatively simple, inexpensive approach to visualize small RNAs without artifacts. However, the radioactive labeling of the probe is sometimes an impediment, especially due to the requirement of specialized facilities. Here we describe a sensitive and simple method to detect and quantify small RNAs using digoxigenin-based nonradioactive RNA blots.

Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecules are currently unknown (Hennelly & Sanbonmatsu, 2012). While chemical probing can reveal single-stranded regions of RNA, traditional approaches to identify sites of chemical modification are time consuming. Mod-seq is a high-throughput method used to map chemical modification sites on RNAs of any size, including complex mixtures of RNA. In this protocol, we describe preparation of Mod-seq high-throughput sequencing libraries from chemically modified RNA. We also describe a software package "Mod-seeker," which is a compilation of scripts written in Python, for the analysis of Mod-seq data. Mod-seeker returns statistically significant modification sites, which can then be used to aid in secondary structure prediction.

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1.

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.

Chemistry enters nucleic acids biology: Enzymatic mechanisms of RNA modification

Modified nucleotides are universally conserved in all living kingdoms and are present in almost all types of cellular RNAs, including tRNA, rRNA, sn(sno)RNA, and mRNA and in recently discovered regulatory RNAs. Altogether, over 110 chemically distinct RNA modifications have been characterized and localized in RNA by various analytical methods. However, this impressive list of known modified nucleotides is certainly incomplete, mainly due to difficulties in identification and characterization of these particular residues in low abundance cellular RNAs. In DNA, modified residues are formed by both enzymatic reactions (like DNA methylations, for example) and by spontaneous chemical reactions resulting from oxidative damage. In contrast, all modified residues characterized in cellular RNA molecules are formed by specific action of dedicated RNA-modification enzymes, which recognize their RNA substrate with high specificity. These RNA-modification enzymes display a great diversity in terms of the chemical reaction and use various low molecular weight cofactors (or co-substrates) in enzymatic catalysis. Depending on the nature of the target base and of the co-substrate, precise chemical mechanisms are used for appropriate activation of the base and the co-substrate in the enzyme active site. In this review, we give an extended summary of the enzymatic mechanisms involved in formation of different methylated nucleotides in RNA, as well as pseudouridine residues, which are almost universally conserved in all living organisms. Other interesting mechanisms include thiolation of uridine residues by ThiI and the reaction of guanine exchange catalyzed by TGT. The latter implies the reversible cleavage of the N-glycosidic bond in order to replace the initially encoded guanine by an aza-guanosine base. Despite the extensive studies of RNA modification and RNA-modification machinery during the last 20 years, our knowledge on the exact chemical steps involved in catalysis of RNA modification remains very limited. Recent discoveries of radical mechanisms involved in base methylation clearly demonstrate that numerous possibilities are used in Nature for these difficult reactions. Future studies are certainly required for better understanding of the enzymatic mechanisms of RNA modification, and this knowledge is crucial not only for basic research, but also for development of new therapeutic molecules.

RNA transport during TMV cell-to-cell movement

Studies during the last 25 years have provided increasing evidence for the ability of plants to support the cell-to-cell and systemic transport of RNA molecules and that this process plays a role in plant development and in the systemic orchestration of cellular responses against pathogens and other environmental challenges. Since RNA viruses exploit the cellular RNA transport machineries for spreading their genomes between cells they represent convenient models to investigate the underlying mechanisms. In this regard, the intercellular spread of Tobacco mosaic virus (TMV) has been studied for many years. The RNA of TMV moves cell-to-cell in a non-encapsidated form in a process depending on virus-encoded movement protein (MP). Here, we discuss the current state of the art in studies using TMV and its MP as a model for RNA transport. While the ability of plants to transport viral and cellular RNA molecules is consistent with RNA transport phenomena in other systems, further studies are needed to increase our ability to visualize viral RNA (vRNA) in vivo and to distinguish RNA-transport related processes from those involved in antiviral defense.

RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function.

The exosome, plugged.

The correct processing, quality control and turnover of cellular RNA molecules is crucial to many aspects of cell physiology. The exosome—a large molecular assembly with exoribonuclease activity—has emerged as a crucial component in many, if not most, stages of RNA metabolism, including degradation and maturation of ribosomal RNA, RNA quality control and turnover of cytosolic messenger RNA (reviewed by Houseley et al , 2006). Exosomes are found in eukaryotes and archaea. The eukaryotic core exosome consists of six polypeptides with sequence similarity to the phosphate‐dependent 3‘→5’ exoribonuclease RNase PH and three protein K homology (KH) and/or S1‐domain‐containing RNA‐binding proteins (Fig 1A; Mitchell et al , 1997). Additional subunits include the RNase R homologue Rrp44/Dis3 (yeast exosome) and the RNase D homologue Rrp6 (nuclear isoform). The archaeal (a) exosome exhibits a simplified subunit composition and is assembled from three copies each of two RNase PH‐like proteins (aRrp41 and aRrp42), aRrp4 or aCsl4 (Evguenieva‐Hackenberg et al , 2003). Figure 1. Comparison of exosomes in archaea and eukaryotes. ( A ) Schematic comparison of subunit composition of archaeal, human and yeast exosomes (viewed from the caps of Rrp4/Rrp40/Csl4). Identified active sites are indicated by black (phosphorolytic) or white (hydrolytic) asterisks. Inactive phosphorolytic subunit pairs are shaded grey. The position of Rrp6 is inferred from native mass spectrometry (Hernandez et al , 2006). Archaeal (a) exosomes might have additional subunits and mixed aRrp4/aCsl4 trimers …

Nucleic acids from intact epithelial cells as a target for stool-based molecular diagnosis of colorectal cancer

Stool-based molecular techniques may improve strategies for colorectal cancer screening. Molecular methods have successfully been applied to detect tumour DNA in stool from patients diagnosed for colorectal carcinoma. In these assays human DNA has to be analyzed against a background of excess nucleic acids from bacteria and dietary waste products. More recently a different diagnostic approach has been described characterizing intact cells isolated from stool. In this study we combine both approaches preparing nucleic acids from isolated epithelial cells to evaluate if: a) tumour cell-specific RNA can be analyzed since cellular RNA molecules are prevented from early digestion by an intact cell membrane; and b) specificity or sensitivity of established DNA-based methods can be improved when epithelial cells are separated from other stool components. Comparing different protocols we found cell isolation using epithelium-specific antibodies to be more effective and reproducible than a technique using density gradient centrifugation. A detection limit of 10(4) cells per ml stool was determined when samples from healthy volunteers were spiked with epithelial cells. Amplification of human sequences from total stool DNA was more efficient than a correspondent amplification of DNA extracted from isolated cells, so that an improvement of DNA-based methods cannot be expected by introducing cell isolation procedures. RNA detection was successful in 1 of 5 patients with confirmed diagnosis of colorectal cancer. The authors suggest that low numbers of detectable cells might rather be a biological than an analytical problem limiting a routinely performed method for colorectal cancer diagnosis.

Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation.

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.

Advances in the Development of Ribozymes and Antisense Oligodeoxynucleotides as Antiviral Agents for Human Papillomaviruses

Urogenital human papillomavirus (HPV) infections are the most common viral sexually transmitted disease in women. On a worldwide basis cervical cancer is the second most prevalent cancer of women. Although HPV infection is not sufficient to induce cancer, the causal relation between high-risk HPV infection and cervical cancer is well established. Over 99% of cervical cancers are positive for high-risk HPV. Therefore, there is a need for newer approaches to treat HPV infection. Two novel approaches for inactivating gene expression involve ribozymes and oligonucleotides. Methods for identification of target genes involved in neoplastic transformation and tumour growth have been established, and these will lead to therapeutic approaches without any damage to normal cellular RNA molecules, which is often associated with conventional therapeutics. Ribozymes and oligonucleotides represent rational antiviral approaches for inhibiting the growth of cervical lesions and carcinomas by interfering with E6/E7 RNA production. The E6 and E7 genes of high-risk HPVs cooperate to immortalize primary epithelial cells and because they are found in cervical cancer are considered the hallmark of cervical cancer. The use and modification of ribozymes and antisense oligodeoxynucleotides can inhibit the growth of HPV-16 and HPV-18 immortalized cells, and tumour cells by eliminating E6/E7 transcript. Hammerhead and hairpin ribozymes have been widely studied because of their potential use for gene therapy and their place as therapeutic tools for cervical cancer is being evaluated. Although antiviral ribozymes and antisense molecules have been effective as in vitro or in vivo inhibitors of high-risk HPV-positive cells, none is currently in clinical trial. There are, however, a number of other antisense therapies in Phase I-III clinical trial for several oncogenes.

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.

Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.

It is commonly accepted that the reverse-transcribed cellular RNA molecules, called retroposons, integrate at staggered breaks in mammalian chromosomes. However, unlike what was previously thought, most of the staggered breaks are not generated by random nicking. One of the two nicks involved is primarily associated with the 5'-TTAAAA hexanucleotide and its variants derived by a single base substitution, particularly A --> G and T --> C. It is probably generated in the antisense strand between the consensus bases 3'-AA and TTTT complementary to 5'-TTAAAA. The sense strand is nicked at variable distances from the TTAAAA consensus site toward the 3' end, preferably within 15-16 base pairs. The base composition near the second nicking site is also nonrandom at positions preceding the nick. On the basis of the observed sequence patterns it is proposed that integration of mammalian retroposons is mediated by an enzyme with endonucleolytic activity. The best candidate for such enzyme may be the reverse transcriptase encoded by the L1 non-long-terminal-repeat retrotransposon, which contains a freshly reported domain homologous to the apurinic/apyrimidinic (AP) endonuclease family [Martin, F., Olivares, M., Lopez, M. C. & Alonso, C. (1996) Trends Biochem. Sci. 21, 283-285; Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916] and shows nicking in vitro with preference for targets similar to 5'-TTAAAA/3'-AATTTT consensus sequence [Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916]. A model for integration of mammalian retroposons based on the presented data is discussed.