Alu Repeats in the Human Genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long and short interspersed elements. An Alu element is a dimer of similar, but not identical, fragments of total size about 300 bp, and originates from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3"-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats constitute more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently have been inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

An unusual Alu repeat sequence within the CAD gene

Summary There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangments may be a factor in generating the diversity of Alu repeats found in primate genomes.     

Phylogenetic affinities of tarsier in the context of primate Alu repeats

Related genomes tend to be colonized by the same or similar repetitive sequence elements. Analysis of these elements provides useful taxonomic information. We have sequenced Alu repeats from tarsier and compared them with those from strepsirhine prosimians (lemurs, sifaka, and galago) and the human genome. Tarsier elements cluster with Alu subfamilies from the human lineage. The oldest subfamily in tarsier and the most abundant human subfamilies share an RNA secondary structure motif which is absent both in the earliest dimeric Alu Jo and in the strepsirhine elements. These findings are consistent with the view that tarsiers form a sister clade with anthropoides rather than with other prosimians. Alu repeats in tarsier genome are relatively old, which indicates a dramatic slowdown or even an arrest of these elements' amplification about 20 Myr ago.     

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. I. Noncanonical structures

We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluS_x sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites—PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alu_bcl2 dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases.     

Invasion of the human albumin-alpha-fetoprotein gene family by Alu, Kpn, and two novel repetitive DNA elements

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.     

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.     

Characterization of the primate-specific repetitive DNA element MER1.

Computer analyses of the 3'-flanking DNA sequence of the human elastase I gene revealed a significant degree of similarity with seven human gene sequences in the GenBank and EMBL databases. Genomic Southern analysis indicates that the shared nucleotide sequences are a primate-specific family of short interspersed elements. These elements are members of MER1 sequences (medium reiteration frequency sequences). The consensus sequence of MER1 repeats spans 543 nucleotides and contains several inverted repeats. Since the copy number of MER1 elements seems to be much smaller than that of Alu and L1 repeats, MER1 elements may provide useful landmarks marks for human genome mapping.     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editing

With over one million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alus that are in opposite orientation gives rise to double-stranded RNA (dsRNA). Although the presence of dsRNA in the cell was previously thought to only occur during viral infection, it is now known that cells express many endogenous small dsRNAs, such as short interfering RNA (siRNAs) and microRNA (miRNAs), which regulate gene expression. It is possible that long dsRNA structures formed from Alu elements influence gene expression. Here, we report that human mRNAs containing inverted Alu elements are present in the mammalian cytoplasm. The presence of these long intramolecular dsRNA structures within 3'-UTRs decreases translational efficiency, and although the structures undergo extensive editing in vivo, the effects on translation are independent of the presence of inosine. As inverted Alus are predicted to reside in >5% of human protein-coding genes, these intramolecular dsRNA structures are important regulators of gene expression.     

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.     

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved

Summary In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.