The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Dicer-dependent biogenesis of small RNAs derived from 7SL RNA

It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting. In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)

Copies of two repetitive elements of the genome of common tree shrew (Tupaia glis) were cloned and sequenced. The first element, Tu III, is a approximately 260 bp long short interspersed element (SINE) with the 5'-end derived from glycine RNA. Tu III carries long polypurine- and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of smooth-tailed tree shrew of another genus (Dendrogale). Tu III seems to be confined to the order Scandentia (tree shrews) since it was not found in DNA of other tested mammals. The second element Tu-SAT1 is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.     

Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)

Copies of two repetitive elements of the common tree shrew (Tupaia glis) genome were cloned and sequenced. The first element, Tu III, is a ～260 bp long short interspersed element (SINE) with the 5′ end derived from glycine RNA. Tu III carries long polypurine-and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of the smooth-tailed tree shrew of another genus (Dendrogale). Tu III appears to be confined to the order Scandentia since it was not found in the DNA of other tested mammals. The second element, Tu-SAT1, is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.     

Transfer RNA-like structure of the human Alu family: Implications of its generation mechanism and possible functions

Summary Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA^Lys, tRNA^Ile, tRNA^Thr, and tRNA^Tyr, which comprise a structurally related family, tRNA^Lys is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. TheGalago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.     

Evolution of secondary structure in the family of 7SL-like RNAs

Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs. These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence. The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species. Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs. To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA.RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey). RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison. Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups. Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes. In RNAs that do not proliferate by retroposition these motifs are modified further. This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.Abbreviations FAM fossil Alu element - FLAM free left Alu monomer - FRAM free right Alu monomer - L-Alu left Alu subunit - R-Alu right Alu subunit Correspondence to: D. LabudaDedicated to Dr. Robert Cedergren on the occasion of his 25th anniversary at the University of Montreal     

Origin of the Alu family: a family of Alu-like monomers gave birth to the left and the right arms of the Alu elements.

The Alu dimeric elements are a common feature of the primate genomes, where they constitute a family of related sequences (1). The identification of a free left Alu monomer (FLAM) family plus a free right Alu monomer (FRAM) family suggests that the dimeric structure results from the fusion of a FLAM sequence with a FRAM sequence (2). Here, we describe a very old Alu-like monomeric family, referred to as FAM for fossil Alu monomer. This family arose from a 7SL RNA sequence and gave birth to the FLAM and FRAM families. From the results obtained, the evolution of the Alu family can be subdivided into two phases. The first phase, which involves only monomeric elements, is characterized by deep remodelling of the progenitor sequences and ends with the appearance of the first Alu dimeric element through the fusion of a FLAM and a FRAM element. The second phase, still in progress, starts with the first Alu dimeric element. This phase is characterized by the stabilization of the progenitor sequences.     

First application of the SINE (short interspersed repetitive element) method to infer phylogenetic relationships in reptiles: an example from the turtle superfamily Testudinoidea

Although turtles (order Testudines) constitute one of the major reptile groups, their phylogenetic relationships remain largely unresolved. Hence, we attempted to elucidate their phylogeny using the SINE (short interspersed repetitive element) method, in which the sharing of a SINE at orthologous loci is indicative of synapomorphy. First, a detailed characterization of the tortoise polIII/SINE was conducted using 10 species from eight families of hidden-necked turtles (suborder Cryptodira). Our analysis of 382 SINE sequences newly isolated in the present study revealed two subgroups, namely Cry I and Cry II, which were distinguishable according to diagnostic nucleotides in the 3' region. Furthermore, four (IA-ID) and five (IIA-IIE) different SINE types were identified within Cry I and Cry II subgroups, respectively, based on features of insertions/deletions located in the middle of the SINE sequences. The relative frequency of occurrence of the subgroups and the types of SINEs in this family were highly variable among different lineages of turtles, suggesting active differential retroposition in each lineage. Further application of the SINE method using the most retrotranspositionally active types, namely IB and IC, challenged the established phylogenetic relationships of Bataguridae and its related families. The data for 11 orthologous loci demonstrated a close relationship between Bataguridae and Testudinidae, as well as the presence of the three clades within Bataguridae. Although the SINE method has been used to infer the phylogenies of a number of vertebrate groups, it has never been applied to reptiles. The present study represents the first application of this method to a phylogenetic analysis of this class of vertebrates, and it provides detailed information on the SINE subgroups and types. This information may be applied to the phylogenetic resolution of relevant turtle lineages.     

Revision of consensus sequence of human Alu repeats--a review

Nucleotide sequences of 50 human Alu repeats and their flanking regions are presented together with the consensus sequence based on the literature and our findings. The results indicate the need for some revisions of the Alu consensus sequence published by Deininger et al. (1981). Most nucleotide substitutions among the Alu members are transitions, rather than transversions. The Alu sequence seems to consist of 'conserved' regions and 'variable' regions. The conserved regions consist of a 25-bp region between nt positions 23 and 47 and a 16-bp region between nt positions 245 and 260. The 16-bp region corresponds to the region of 7SL RNA that is claimed to fold and become paired with the internal promoter sequence. Two A-rich regions, one located at the right end of the first monomer and the other at the right end of the second monomer, are variable. No defined property was found with direct repeats flanking the Alu repeats.     

Human 7SL RNA consists of a 140 nucleotide middle-repetitive sequence inserted in an alu sequence

We have cloned and sequenced a cDNA copy of in vitro-polyadenylated 7SL RNA of HeLa cells. The cloned fragment is 303 bp long and has a composite structure. A central block of 140 bp is homologous to a new set of human middle-repetitive sequences. This block appears to be inserted in an Alu consensus sequence, 100 bp from the 5' end and 40 bp from the 3' end of the Alu monomer. Two 6 bp direct repeats are found at the junction between the Alu flanking sequences and the central element. The analysis of several clones shows the existence of sequence microheterogeneity in the 5' portion of the molecule. The 7L DNA probably represents a subset of the Alu family of DNA, highly conserved in evolution.     

RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements

While the significance of middle repetitive elements had been neglected for a long time, there are again tendencies to ascribe most members of a given middle repetitive sequence family a functional role--as if the discussion of SINE (short interspersed repetitive elements) function only can occupy extreme positions. In this article, I argue that differences between the various classes of retrosequences concern mainly their copy numbers. Consequently, the function of SINEs should be viewed as pragmatic such as, for example, mRNA-derived retrosequences, without underestimating the impact of retroposition for generation of novel protein coding genes or parts thereof (exon shuffling by retroposition) and in particular of SINEs (and retroelements) in modulating genes and their expression. Rapid genomic change by accumulating retrosequences may even facilitate speciation [McDonald, J.F., 1995. Transposable elements: possible catalysts of organismic evolution. Trends Ecol. Evol. 10, 123-126.] In addition to providing mobile regulatory elements, small RNA-derived retrosequences including SINEs can, in analogy to mRNA-derived retrosequences, also give rise to novel small RNA genes. Perhaps not representative for all SINE/master gene relationships, we gained significant knowledge by studying the small neuronal non-messenger RNAs, namely BC1 RNA in rodents and BC200 RNA in primates. BC1 is the first identified master gene generating a subclass of ID repetitive elements, and BC200 is the only known Alu element (monomeric) that was exapted as a novel small RNA encoding gene.     

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.     

5S rRNA-derived and tRNA-derived SINEs in fruit bats

Most short retroposons (SINEs) descend from cellular tRNA of 7SL RNA. Here, four new SINEs were found in megabats (Megachiroptera) but neither in microbats nor in other mammals. Two of them, MEG-RS and MEG-RL, descend from another cellular RNA, 5S rRNA; one (MEG-T2) is a tRNA-derived SINE; and MEG-TR is a hybrid tRNA/5S rRNA SINE. Insertion locus analysis suggests that these SINEs were active in the recent fruit bat evolution. Analysis of MEG-RS and MEG-RL in comparison with other few 5S rRNA-derived SINEs demonstrates that the internal RNA polymerase III promoter is their most invariant region, while the secondary structure is more variable. The mechanisms underlying the modular structure of these and other SINEs as well as their variation are discussed. The scenario of evolution of MEG SINEs is proposed.     

Conservation of sequences in related genomes of Apodemus: constraints on the maintenance of satellite DNA sequences.

Satellites from two related species of the Apodemus genus, A. sylvaticus and A. flavicollis, have been analysed with restriction enzymes Taq I, Alu I and Hind III. The restriction maps are closely conserved between species and show a novel feature of two differing internal periodicities within a 375 base pair repeating unit detected by two different restriction enzymes. This places constraints on the introduction of the observed restriction sites according to current models such as unequal crossing-over. The implications of such a conserved sequence and its presence in other species are discussed.