Showing papers on "Alu element published in 2009"

Mobile elements create structural variation: analysis of a complete human genome.

Abstract: Structural variants (SVs) are common in the human genome. Because approximately half of the human genome consists of repetitive, transposable DNA sequences, it is plausible that these elements play an important role in generating SVs in humans. Sequencing of the diploid genome of one individual human (HuRef) affords us the opportunity to assess, for the first time, the impact of mobile elements on SVs in an individual in a thorough and unbiased fashion. In this study, we systematically evaluated more than 8000 SVs to identify mobile element-associated SVs as small as 100 bp and specific to the HuRef genome. Combining computational and experimental analyses, we identified and validated 706 mobile element insertion events (including Alu, L1, SVA elements, and nonclassical insertions), which added more than 305 kb of new DNA sequence to the HuRef genome compared with the Human Genome Project (HGP) reference sequence (hg18). We also identified 140 mobile element-associated deletions, which removed ~126 kb of sequence from the HuRef genome. Overall, ~10% of the HuRef-specific indels larger than 100 bp are caused by mobile element-associated events. More than one-third of the insertion/deletion events occurred in genic regions, and new Alu insertions occurred in exons of three human genes. Based on the number of insertions and the estimated time to the most recent common ancestor of HuRef and the HGP reference genome, we estimated the Alu, L1, and SVA retrotransposition rates to be one in 21 births, 212 births, and 916 births, respectively. This study presents the first comprehensive analysis of mobile element-related structural variants in the complete DNA sequence of an individual and demonstrates that mobile elements play an important role in generating inter-individual structural variation.

Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer.

Abstract: LINE-1 and Alu elements are non-LTR retrotransposons, constituting together over 30% of the human genome and they are frequently hypomethylated in human tumors A relationship between global hypomethylation and genomic instability has been shown, however, there is little evidence to suggest active role for hypomethylation-mediated reactivation of retroelements in human cancer In our study, we examined by Pyrosequencing the methylation levels of LINE-1 and Alu sequences in 48 primary nonsmall cell carcinomas and their paired adjacent tissues We demonstrate a significant reduction of the methylation levels of both elements (p = 77 x 10(-14) and 96 x 10(-7), respectively) The methylation indices of the 2 elements correlated (p = 0006), suggesting a possible common mechanism for their methylation maintenance Genomic instability was measured utilizing 11 fluorescent microsatellite markers located on lung cancer hot-spot regions such as 3p, 5q 9p, 13q and 17p Hypomethylation of both transposable elements was associated with increased genomic instability (LINE, p = 71 x 10(-5); Alu, p = 0008) The reduction of the methylation index of LINE-1 and Alu following treatment of 3 lung cell lines with 5-aza-2'-deoxycitidine, consistently resulted in increased expression of both elements Our study demonstrates the strong link between hypomethylation of transposable elements with genomic instability in non-small cell lung cancer and provides early evidence for a potential active role of these elements in lung neoplasia As demethylating agents are now entering lung cancer trials, it is imperative to gain a greater insight into the potential reactivation of silent retrotransposons in order to advance for the clinical utilization of epigenetics in cancer therapy

C19MC microRNAs are processed from introns of large Pol-II, non-protein-coding transcripts

Abstract: MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

Chromatin remodeling at Alu repeats by epigenetic treatment activates silenced microRNA-512-5p with downregulation of Mcl-1 in human gastric cancer cells

Abstract: Epigenetic therapy using DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors has clinical promise for the treatment of human malignancies. To investigate roles of microRNAs (miRNAs) on epigenetic therapy of gastric cancer, the miRNA expression profile was analysed in human gastric cancer cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR) and 4-phenylbutyric acid (PBA). miRNA microarray analysis shows that most of miRNAs activated by 5-Aza-CdR and PBA in gastric cancer cells are located at Alu repeats on chromosome 19. Analyses of chromatin modification show that DNA demethylation and HDAC inhibition at Alu repeats activates silenced miR-512-5p by RNA polymerase II. In addition, activation of miR-512-5p by epigenetic treatment induces suppression of Mcl-1, resulting in apoptosis of gastric cancer cells. These results suggest that chromatin remodeling at Alu repeats plays critical roles in the regulation of miRNA expression and that epigenetic activation of silenced Alu-associated miRNAs could be a novel therapeutic approach for gastric cancer.

Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability

Abstract: Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.

Changes in DNA methylation of tandem DNA repeats are different from interspersed repeats in cancer

Abstract: Hypomethylation of DNA repetitive elements is a common finding in cancer, but very little is known about the DNA methylation changes of different types of DNA repetitive elements, such as interspersed repeats (LINE1 and Alu Yb8) and tandem repeats (Sat-alpha, NBL-2 and D4Z4). We used bisulfite-PCR Pyrosequencing to quantitatively measure the DNA methylation of five different DNA repetitive elements in normal tissue and cancer. In all we studied 10 different tissues from four individuals undergoing autopsy, 34 paired normal and tumor tissues from patients with bladder cancer, 58 patients with chronic myelogenous leukemia and 23 patients with acute promyelocytic leukemia. We found that the DNA methylation of interspersed repeats (LINE1 and Alu Yb8) was very consistent from person to person and tissue to tissue while tandem DNA repeats appeared more variable in normal tissues. In bladder cancer we found clear hypomethylation of LINE1, Alu Yb8, Sat-alpha and NBL-2. Conversely, we found an increase in the DNA methylation levels of D4Z4 from normal to cancer. In contrast leukemia showed no significant changes in the DNA methylation of LINE1 and Alu Yb8, but DNA methylation increases in NBL-2 and D4Z4 tandem repeats. Our findings show that the changes in DNA methylation levels of individual DNA repetitive elements are unique for each repetitive element, which may reflect distinct epigenetic factors and may have important implications in the use of DNA methylation of repetitive elements as global DNA methylation biomarkers.

LINE dancing in the human genome: transposable elements and disease

Abstract: Transposable elements (TEs) have been consistently underestimated in their contribution to genetic instability and human disease. TEs can cause human disease by creating insertional mutations in genes, and also contributing to genetic instability through non-allelic homologous recombination and introduction of sequences that evolve into various cis-acting signals that alter gene expression. Other outcomes of TE activity, such as their potential to cause DNA double-strand breaks or to modulate the epigenetic state of chromosomes, are less fully characterized. The currently active human transposable elements are members of the non-LTR retroelement families, LINE-1, Alu (SINE), and SVA. The impact of germline insertional mutagenesis by TEs is well established, whereas the rate of post-insertional TE-mediated germline mutations and all forms of somatic mutations remain less well quantified. The number of human diseases discovered to be associated with non-allelic homologous recombination between TEs, and particularly between Alu elements, is growing at an unprecedented rate. Improvement in the technology for detection of such events, as well as the mounting interest in the research and medical communities in resolving the underlying causes of the human diseases with unknown etiology, explain this increase. Here, we focus on the most recent advances in understanding of the impact of the active human TEs on the stability of the human genome and its relevance to human disease.

Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediated innate immune response in humans and primates

Abstract: Background About 45% of the human genome is comprised of mobile transposable elements or "junk DNA". The exaptation or co-option of these elements to provide important cellular functions is hypothesized to have played a powerful force in evolution; however, proven examples are rare. An ancient primate-specific Alu short interspersed element (SINE) put the human CAMP gene under the regulation of the vitamin D pathway by providing a perfect vitamin D receptor binding element (VDRE) in its promoter. Subsequent studies demonstrated that the vitamin D-cathelicidin pathway may be a key component of a novel innate immune response of human to infection. The lack of evolutionary conservation in non-primate mammals suggested that this is a primate-specific adaptation. Evidence for evolutionary conservation of this regulation in additional primate lineages would provide strong evidence that the TLR2/1-vitamin D-cathelicidin pathway evolved as a biologically important immune response mechanism protecting human and non-human primates against infection.

5'-transducing SVA retrotransposon groups spread efficiently throughout the human genome

Abstract: SVA elements are nonautonomous non–long terminal repeat (non-LTR) retrotransposons that originated <25 million years ago (Mya) and represent the youngest retrotransposon family in primates. So far their copy number has increased to roughly 3000 in the human genome (Ostertag et al. 2003; Wang et al. 2005). Their ongoing activity in humans sporadically causes disease by randomly inserting into genes (for review, see Belancio et al. 2008). Considering the number of disease-causing insertions relative to their overall copy number, SVA elements are thought to represent a highly active retrotransposon family in humans (Ostertag et al. 2003; Wang et al. 2005). SVA elements stand out from the group of human non-LTR retrotransposons due to their composite structure, including modules derived from other primate repetitive elements. Starting at the 5′-end, a full-length SVA element is composed of a (CCCTCT)n hexamer repeat region; an Alu-like region consisting of three antisense Alu fragments adjacent to an additional sequence of unknown origin; a variable number of tandem repeats (VNTR) region, which is made up of copies of a 36- to 42-bp sequence or of a 49- to 51-bp sequence (Ostertag et al. 2003); and a short interspersed element of retroviral origin (SINE-R) region. The latter is derived from the 3′-end of the env gene and the 3′-LTR of the endogenous retrovirus HERVK-10 (Ono et al. 1987). A poly(A) tail is positioned downstream of the predicted conserved polyadenylation signal AATAAA (Ostertag et al. 2003). The origin of SVA elements can be traced back to the beginnings of hominid primate evolution, only about 18–25 Mya. Their very young evolutionary age represents a unique opportunity to study the entire evolutionary history of a human retrotransposon. In addition, SVA elements may be valuable as markers for primate or human phylogenetic and population genetic studies, as has been the case for Alu elements (Bamshad et al. 2003; Watkins et al. 2003; Xing et al. 2007). In order to draw conclusions on both the assembly process that led to the formation of SVA elements and the mechanism of their transcriptional regulation, we started out by assessing the different structural variants of SVA elements on a single chromosome. Thorough investigation of a single chromosome allows detailed structural inspection of individual elements. We chose chromosome 19 for the following reasons: (1) it is one of the best characterized single human chromosomes with regard to DNA sequence and biology (Grimwood et al. 2004), and (2) with an average density of one element per 0.62 Mb, it was reported to have the highest density of SVA insertions among human chromosomes (Wang et al. 2005). (3) Nearly 55% of this chromosome consists of repetitive elements (Grimwood et al. 2004), whereas the genome average is only 44.8% (Lander et al. 2001). This difference is due mainly to an unusually high content of short interspersed elements (SINEs) on this chromosome. Therefore, we reasoned that representatives of all human SVA variants should also be present on chromosome 19. The comprehensive analysis of chromosome 19 uncovered 116 SVA elements that could be assorted into seven types of structural variants. Novel variants identified were 3′-truncated elements and a significant number of SVA elements with 5′-flanking sequence transductions. Our genome-wide analysis uncovered 220 5′-transducing SVA elements that originated from 93 diverse genomic source loci. The 93 resultant 5′ transduction groups are composed of one to 84 members. The organization of these elements demonstrates that SVA elements can recruit external heterologous promoters for their own transcriptional regulation, which in turn might affect mobilization frequencies. In a number of cases, 5′-transduced sequences are constituted by spliced cellular mRNAs. Therefore, SVA-mediated 5′ transduction represents a further mechanism of retrotransposon-mediated exon shuffling. SVA-driven transduction of 5′-flanking DNA is likely to be one mechanism of genome evolution via increasing genome plasticity and facilitating new combinations of coding and regulatory sequences. Acquisition of new 5′ sequences may also be a common theme in SVA evolution, with the objective of identifying new genomic components whose incorporation increases the efficiency of SVA mobilization.

Alu‐Alu recombination underlies the vast majority of large VHL germline deletions: Molecular characterization and genotype–phenotype correlations in VHL patients

Abstract: Von Hippel-Lindau disease (VHL) is an autosomal dominant cancer syndrome. Affected individuals are predisposed to multiple tumors, primarily of the central nervous system (CNS), eyes, adrenals, and kidneys. The VHL tumor suppressor gene on chromosome 3p26-25 is partially or completely deleted in 20 to 30% of families with VHL. We identified deletions ranging from 0.5 kb to 250 kb affecting part of or the entire VHL and flanking genes in 54 families. In 33 of the index patients, the breakpoints were precisely characterized by DNA sequencing. Of the 66 breakpoints, 90% were located in Alu elements, revealing Alu-mediated recombination as the major mechanism for large germline deletions of the VHL gene, which lies in a region of high Alu density. Interestingly, an AluYa5 element in VHL intron 2, the evolutionarily youngest Alu element and the only such element in the entire region, was found to be the most recombinogenic, involved in 7 out of the 33 deletions. In comparison to VHL patients in general, the 54 index cases and their affected relatives showed a higher occurrence of renal cell carcinomas (RCC) and of CNS hemangioblastomas. We not only noted the association of RCC with retention of the HSPC300 gene, but also observed a significant correlation between retention of HSPC300 and the development of retinal angiomas (AR). This study reveals that germline VHL deletions provide a particularly rich source for the study of Alu-mediated unequal crossover events, and provides evidence for a protective role of the loss of the actin-regulator gene HSPC300 for the development of both RCC and AR.

Evidence for co-evolution between human microRNAs and Alu-repeats.

Abstract: This paper connects Alu repeats, the most abundant repetitive elements in the human genome and microRNAs, small RNAs that alter gene expression at the post-transcriptional level. Base-pair complementarity could be demonstrated between the seed sequence of a subset of human microRNAs and Alu repeats that are integrated parallel (sense) in mRNAs. The most common target site coincides with the evolutionary most conserved part of Alu. A primate-specific gene cluster on chromosome 19 encodes the majority of miRNAs that target the most conserved sense Alu site. The individual miRNA genes within this cluster are flanked by an Alu-LINE signature, which has been duplicated with the clustered miRNA genes. Gene duplication events in this locus are supported by comparing repeat length variations of the LINE elements within the cluster with those in the rest of the chromosome. Thus, a dual relationship exists between an evolutionary young miRNA cluster and their Alu targets that may have evolved in the same time window. One hypothesis for this dual relationship is that these miRNAs could protect against too high rates of duplicative transposition, which would destroy the genome.

Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution

Abstract: Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15–18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.

High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum

Abstract: DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5′ flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer.

Nuclear translocated Ehrlichia chaffeensis ankyrin protein interacts with a specific adenine-rich motif of host promoter and intronic Alu elements

Abstract: Ehrlichiae are obligately intracellular bacteria that reside and replicate in phagocytes by circumventing host cell defenses and modulating cellular processes, including host cell gene transcription. However, the mechanisms by which ehrlichiae influence host gene transcription have largely remained undetermined. Numerous ankyrin and tandem repeat-containing proteins associated with host-pathogen interactions have been identified in Ehrlichia species, but their roles in pathobiology are unknown. In this study, we determined by confocal immunofluorescence microscopy and by immunodetection in purified nuclear extracts that the ankyrin repeat-containing protein p200 is translocated to the nuclei of Ehrlichia-infected monocytes. Chromatin immunoprecipitation (ChIP) with DNA sequencing revealed an Ehrlichia chaffeensis p200 interaction located within host promoter and intronic Alu-Sx elements, the most abundant repetitive elements in the human genome. A specific adenine-rich (mid-A-stretch) motif within Alu-Sx elements was identified using electrophoretic mobility shift and NoShift assays. Whole-genome analysis with ChIP and DNA microarray analysis (ChIP-chip) determined that genes (n = 456) with promoter Alu elements primarily related to transcription, apoptosis, ATPase activity, and structural proteins associated with the nucleus and membrane-bound organelles were the primary targets of p200. Several p200 target genes (encoding tumor necrosis factor alpha, Stat1, and CD48) associated with ehrlichial pathobiology were strongly upregulated during infection, as determined by quantitative PCR. This is the first study to identify a nuclear translocation of bacterially encoded protein by E. chaffeensis and to identify a specific binding motif and genes that are primary targets of a novel molecular strategy to reprogram host cell gene expression to promote survival of the pathogen.

Alu and b1 repeats have been selectively retained in the upstream and intronic regions of genes of specific functional classes.

Abstract: Alu and B1 repeats are mobile elements that originated in an initial duplication of the 7SL RNA gene prior to the primate-rodent split about 80 million years ago and currently account for a substantial fraction of the human and mouse genome, respectively. Following the primate-rodent split, Alu and B1 elements spread independently in each of the two genomes in a seemingly random manner, and, according to the prevailing hypothesis, negative selection shaped their final distribution in each genome by forcing the selective loss of certain Alu and B1 copies. In this paper, contrary to the prevailing hypothesis, we present evidence that Alu and B1 elements have been selectively retained in the upstream and intronic regions of genes belonging to specific functional classes. At the same time, we found no evidence for selective loss of these elements in any functional class. A subset of the functional links we discovered corresponds to functions where Alu involvement has actually been experimentally validated, whereas the majority of the functional links we report are novel. Finally, the unexpected finding that Alu and B1 elements show similar biases in their distribution across functional classes, despite having spread independently in their respective genomes, further supports our claim that the extant instances of Alu and B1 elements are the result of positive selection.

Using Alu Elements as Polyadenylation Sites: A Case of Retroposon Exaptation

Abstract: Of the 1.1 million Alu retroposons in the human genome, about 10,000 are inserted in the 3' untranslated regions (UTR) of protein-coding genes and 1% of these (107 events) are active as polyadenylation sites (PASs). Strikingly, although Alu's in 3' UTR are indifferently inserted in the forward or reverse direction, 99% of polyadenylation-active Alu sequences are forward oriented. Consensus Alu+ sequences contain sites that can give rise to polyadenylation signals and enhancers through a few point mutations. We found that the strand bias of polyadenylation-active Alu's reflects a radical difference in the fitness of sense and antisense Alu's toward cleavage/polyadenylation activity. In contrast to previous beliefs, Alu inserts do not necessarily represent weak or cryptic PASs; instead, they often constitute the major or the unique PAS in a gene, adding to the growing list of Alu exaptations. Finally, some Alu-borne PASs are intronic and produce truncated transcripts that may impact gene function and/or contribute to gene remodeling.

Diverse cis factors controlling Alu retrotransposition: What causes Alu elements to die?

Abstract: The human genome contains nearly 1.1 million Alu elements comprising roughly 11% of its total DNA content. Alu elements use a copy and paste retrotransposition mechanism that can result in de novo disease insertion alleles. There are nearly 900,000 old Alu elements from subfamilies S and J that appear to be almost completely inactive, and about 200,000 from subfamily Y or younger, which include a few thousand copies of the Ya5 subfamily which makes up the majority of current activity. Given the much higher copy number of the older Alu subfamilies, it is not known why all of the active Alu elements belong to the younger subfamilies. We present a systematic analysis evaluating the observed sequence variation in the different sections of an Alu element on retrotransposition. The length of the longest number of uninterrupted adenines in the A-tail, the degree of A-tail heterogeneity, the length of the 3′ unique end after the A-tail and before the RNA polymerase III terminator, and random mutations found in the right monomer all modulate the retrotransposition efficiency. These changes occur over different evolutionary time frames. The combined impact of sequence changes in all of these regions explains why young Alus are currently causing disease through retrotransposition, and the old Alus have lost their ability to retrotranspose. We present a predictive model to evaluate the retrotransposition capability of individual Alu elements and successfully applied it to identify the first putative source element for a disease-causing Alu insertion in a patient with cystic fibrosis.

Comparative analysis of Alu repeats in primate genomes

Abstract: Using bacteria artificial chromosome (BAC) end sequences (16.9 Mb) and high-quality alignments of genomic sequences (17.4 Mb), we performed a global assessment of the divergence distributions, phylogenies, and consensus sequences for Alu elements in primates including lemur, marmoset, macaque, baboon, and chimpanzee as compared to human. We found that in lemurs, Alu elements show a broader and more symmetric sequence divergence distribution, suggesting a steady rate of Alu retrotransposition activity among prosimians. In contrast, Alu elements in anthropoids show a skewed distribution shifted toward more ancient elements with continual declining rates in recent Alu activity along the hominoid lineage of evolution. Using an integrated approach combining mutation profile and insertion/deletion analyses, we identified nine novel lineage-specific Alu subfamilies in lemur (seven), marmoset (one), and baboon/macaque (one) containing multiple diagnostic mutations distinct from their human counterparts—Alu J, S, and Y subfamilies, respectively. Among these primates, we show that that the lemur has the lowest density of Alu repeats (55 repeats/Mb), while marmoset has the greatest abundance (188 repeats/Mb). We estimate that ∼70% of lemur and 16% of marmoset Alu elements belong to lineage-specific subfamilies. Our analysis has provided an evolutionary framework for further classification and refinement of the Alu repeat phylogeny. The differences in the distribution and rates of Alu activity have played an important role in subtly reshaping the structure of primate genomes. The functional consequences of these changes among the diverse primate lineages over such short periods of evolutionary time are an important area of future investigation.

The RNA polymerase dictates ORF1 requirement and timing of LINE and SINE retrotransposition.

Abstract: Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners—such as the retropseudogenes, SVA, and the SINE, Alu—are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the “pol II Alu transcript” behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery

Abstract: Despite decades of research, the question of how the mRNA splicing machinery precisely identifies short exonic islands within the vast intronic oceans remains to a large extent obscure. In this study, we analyzed Alu exonization events, aiming to understand the requirements for correct selection of exons. Comparison of exonizing Alus to their non-exonizing counterparts is informative because Alus in these two groups have retained high sequence similarity but are perceived differently by the splicing machinery. We identified and characterized numerous features used by the splicing machinery to discriminate between Alu exons and their non-exonizing counterparts. Of these, the most novel is secondary structure: Alu exons in general and their 5' splice sites (5'ss) in particular are characterized by decreased stability of local secondary structures with respect to their non-exonizing counterparts. We detected numerous further differences between Alu exons and their non-exonizing counterparts, among others in terms of exon-intron architecture and strength of splicing signals, enhancers, and silencers. Support vector machine analysis revealed that these features allow a high level of discrimination (AUC = 0.91) between exonizing and non-exonizing Alus. Moreover, the computationally derived probabilities of exonization significantly correlated with the biological inclusion level of the Alu exons, and the model could also be extended to general datasets of constitutive and alternative exons. This indicates that the features detected and explored in this study provide the basis not only for precise exon selection but also for the fine-tuned regulation thereof, manifested in cases of alternative splicing.

Alu-directed transcriptional regulation of some novel miRNAs.

Abstract: Despite many studies on the biogenesis, molecular structure and biological functions of microRNAs, little is known about the transcriptional regulatory mechanisms controlling the spatiotemporal expression pattern of human miRNA gene loci. Several lines of experimental results have indicated that both polymerase II (Pol-II) and polymerase III (Pol-III) may be involved in transcribing miRNAs. Here, we assessed the genomic evidence for Alu-directed transcriptional regulation of some novel miRNA genes in humans. Our data demonstrate that the expression of these Alu-related miRNAs may be modulated by Pol-III. We present a comprehensive exploration of the Alu-directed transcriptional regulation of some new miRNAs. Using a new computational approach, a variety of Alu-related sequences from multiple sources were pooled and filtered to obtain a subset containing Alu elements and characterized miRNA genes for which there is clear evidence of full-length transcription (embedded in EST). We systematically demonstrated that 73 miRNAs including five known ones may be transcribed by Pol-III through Alu or MIR. Among the new miRNAs, 33 were determined by high-throughput Solexa sequencing. Real-time TaqMan PCR and Northern blotting verified that three newly identified miRNAs could be induced to co-express with their upstream Alu transcripts by heat shock or cycloheximide. Through genomic analysis, Solexa sequencing and experimental validation, we have identified candidate sequences for Alu-related miRNAs, and have found that the transcription of these miRNAs could be governed by Pol-III. Thus, this study may elucidate the mechanisms by which the expression of a class of small RNAs may be regulated by their upstream repeat elements.

Evidence for large diversity in the human transcriptome created by Alu RNA editing

Abstract: Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome.

Alu repeats increase local recombination rates

Abstract: Recombination rates vary widely across the human genome, but little of that variation is correlated with known DNA sequence features. The genome contains more than one million Alu mobile element insertions, and these insertions have been implicated in non-homologous recombination, modulation of DNA methylation, and transcriptional regulation. If individual Alu insertions have even modest effects on local recombination rates, they could collectively have a significant impact on the pattern of linkage disequilibrium in the human genome and on the evolution of the Alu family itself. We carried out sequencing, SNP identification, and SNP genotyping around 19 AluY insertion loci in 347 individuals sampled from diverse populations, then used the SNP genotypes to estimate local recombination rates around the AluY loci. The loci and SNPs were chosen so as to minimize other factors (such as SNP ascertainment bias and SNP density) that could influence recombination rate estimates. We detected a significant increase in recombination rate within ~2 kb of the AluY insertions in our African population sample. To test this observation against a larger set of AluY insertions, we applied our locus- and SNP-selection design and analyses to the HapMap Phase II data. In that data set, we observed a significantly increased recombination rate near AluY insertions in both the CEU and YRI populations. We show that the presence of a fixed AluY insertion is significantly predictive of an elevated local recombination rate within 2 kb of the insertion, independent of other known predictors. The magnitude of this effect, approximately a 6% increase, is comparable to the effects of some recombinogenic DNA sequence motifs identified via their association with recombination hot spots.

Rare mutations of FGFR2 causing apert syndrome: identification of the first partial gene deletion, and an Alu element insertion from a new subfamily†

Abstract: Apert syndrome (AS) is a severe disorder, characterized by craniosynostosis and complex syndactyly of the hands and feet. Two heterozygous gain-of-function substitutions (Ser252Trp and Pro253Arg) in exon IIIa of fibroblast growth factor receptor 2 (FGFR2) are responsible for >98% of cases. Here we describe two novel mutations in FGFR2 in the two patients in whom a mutation had not previously been found in our cohort of 227 AS cases. The first is a 1.93-kb deletion, removing exon IIIc and substantial portions of the flanking introns. This is the first large FGFR2 deletion described in any individual with craniosynostosis. The other mutation is a 5' truncated Alu insertion into exon IIIc. This is the third Alu insertion identified in AS; all have occurred within an interval of only 104 bp, representing an enrichment of over a million-fold compared to the background genomic rate. We show that the inserted Alu element belongs to a small subfamily, not previously known to be mobile, which we term Alu Yk13. Both the deletion and insertion are likely to act by a similar gain-of-function mechanism in which disruption of exon IIIc leads to illegitimate mesenchymal expression of an FGFR2 spliceform containing the alternatively spliced exon IIIb. All the AS-associated Alu insertions have arisen in the paternal germline; we propose that their enrichment in FGFR2 is driven by positive selection of the mutant spermatogonial progenitors, a mechanism analogous to that explaining why the canonical AS nucleotide substitutions also reach exceptionally high levels in sperm.

A novel protein isoform of the multicopy human NAIP gene derives from intragenic Alu SINE promoters.

Abstract: The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene.