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.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

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 (LINEs) and short (SINEs) interspersed elements. Alu elements are each a dimer of similar, but not identical, fragments of total size about 300 bp, and originate 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 comprise 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 inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.     

Alu repeats and human genomic diversity

During the past 65 million years, Alu elements have propagated to more than one million copies in primate genomes, which has resulted in the generation of a series of Alu subfamilies of different ages. Alu elements affect the genome in several ways, causing insertion mutations, recombination between elements, gene conversion and alterations in gene expression. Alu-insertion polymorphisms are a boon for the study of human population genetics and primate comparative genomics because they are neutral genetic markers of identical descent with known ancestral states.     

Phylogenetic roots of Alu-mediated rearrangements leading to cancer.

There are over a million Alu repetitive elements dispersed throughout the human genome, and a high level of Alu-sequence similarity ensures a strong propensity for unequal crossover events, some of which have lead to deleterious oncogenic rearrangements. Furthermore, Alu insertions introduce consensus 3' splice sites, which potentially facilitate alternative splicing. Not surprisingly, Alu-mediated defective splicing has also been associated with cancer. To investigate a possible correlation between the expansion of Alu repeats associated with primate divergence and predisposition to cancer, 4 Alu-mediated rearrangements--known to be the basis of cancer--were selected for phylogenetic analysis of the necessary genotype. In these 4 cases, it was determined that the different phylogenetic age of the oncogenic recombination-prone genotype reflected the evolutionary history of Alu repeats spreading to new genomic sites. Our data implies that the evolutionary expansion of Alu repeats to new genomic locations establishes new predispositions to cancer in various primate species.     

A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution.

Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C-terminal variants of SRP14 in higher primates.     

Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A

Short interspersed elements, such as Alu elements, have propagated to more than one million copies in the human genome. They affect the genome in several ways, caused by retrotransposition, recombination between elements, gene conversion, and alterations in gene expression. These events, including novel insertions into active genes, have been associated with a number of human disorders. Hemophilia A is an X-linked severe bleeding disorder and is caused by mutations in the Factor VIII gene. The spectrum of mutations includes point mutations, rearrangements, insertions, and deletions. Recently, an Alu retrotransposition event in a coding exon has been reported in a family with a severe form of hemophilia A. This was the first report of an Alu insertion in the Factor VIII gene. Here, we report a second Alu insertion event that lies in an intron of the same gene that causes exon skipping and the complete disruption of gene expression.     

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.     

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.