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.     

Characterization of Novikoff hepatoma small RNAs homologous to repetitive DNAs

Three minor small RNA species from Novikoff hepatoma cells, with homology to repetitive DNA sequences, have been identified and characterized. These small RNAs, designated 5.1S, 6S and T3 RNAs, show homology to Alu 1, Alu 2, and Alu 3 sequences, respectively. 6S and T3 RNAs were found both in the nucleus and cytoplasm, whereas 5.1S RNA was not found in the nucleus. Neural tissues were found to contain a 6S-sized BC1 RNA with homology to I.D. sequences [19]; in contrast, the current study shows that Novikoff hepatoma cells contain a 75–80 nucleotide long (T3) RNA, homologous to I.D. sequences. These data suggest that BCl and T3 small RNAs, homologous to I.D. sequences, are expressed in a tissue-specific manner. These results also show that in addition to the abundant 7SL, 4.5S and 4.5S1 RNAs having homology to repetitive DNA, Novikoff hepatoma cells also contain several minor small RNAs with homology to repetitive sequences.     

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.     

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.     

Emergence of master sequences in families of retroposons derived from 7sl RNA

The past few years have brought new insight into the evolution of families of retroposons. These are composed of a very small number of master sequences able to duplicate, and a large majority of copies that are inactive for retroposition. During the course of time, successive replacements of master sequences have produced waves of amplification that are recognizable as subfamilies. In the Alu and the B1 families, one can distinguish two evolutionary periods. The first involves only monomeric elements that are now extinguished (fossil elements) and is characterized by deep remodeling of the sequences. This period ends, in primates, with the fusion of a free left and a free right Alu monomer, producing the first modern Alu dimeric element; in rodents it ends with a tandem duplication of 29 bp to create the first modern B1 element. The second period is characterized by a great stability of the master sequences. The observed turn-over of master sequences is still an enigma. However, analysis of the contemporary master sequences and of the oldest master sequences provide some clues. Here, we review the very first stages of the appearance of the Alu and the B1 families in mammalian genomes.     

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.     

Characterization of novel Alu- and tRNA-related SINEs from the tree shrew and evolutionary implications of their origins

We characterized two novel 7SL RNA-derived short interspersed nuclear element (SINE) families (Tu types I and II) and a novel tRNA-derived SINE family (Tu type III) from the tree shrew (Tupaia belangeri). Tu type I contains a monomer unit of a 7SL RNA-derived Alu-like sequence and a tRNA-derived region that includes internal RNA polymerase III promoters. Tu type II has a similar hybrid structure, although the monomer unit of the 7SL RNA-derived sequence is replaced by a dimer. Along with the primate Alu, the galago Alu type II, and the rodent B1, these two families represent the fourth and fifth 7SL RNA-derived SINE families to be identified. Furthermore, comparison of the Alu domains of Tu types I and II with those of other 7SL RNA-derived SINEs reveals that the nucleotides responsible for stabilization of the Alu domain have been conserved during evolution, providing the possibility that these conserved nucleotides play an indispensable role in retropositional activity. Evolutionary relationships among these 7SL RNA-derived SINE families, as well as phylogenetic relationships of their host species, are discussed.     

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.     

Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.     

Various aspects of evolution of Alu repeats in mammals

A complex study on various evolutionary peculiarities of the mammalia dispersed Alu repeats (Alu repeats of primates and B1 of rodents) has been carried out by phylogenetic analysis. A phylogenetic tree, containing the 7SL RNA genes and the Alu repeats of primates and rodents has been constructed. It has been shown that the branch of the phyletic line leading to the Alu repeats of primates and B1 of rodents from the 7SL RNA genes occurred after the divergence of the 7SL RNA genes of amphibia and mammalia, but before the divergence of the 7SL RNA genes of primates and rodents (250.10 years ago). A statistically reliable slowing down in the evolutionary rates of one of two monomers for the human Alu repeats has been proved. It may be caused by the functional load of the corresponding monomer in connection with the presence of a definit regulatory site in it.     

Computer analysis of the sequence relationships among 4.5S RNA molecular species from various sources

Nucleotide sequence homology among 4.5S RNAs from various organisms was examined by computer analysis to evaluate their sequence relationships. Chloroplast 4.5S rRNAs of wheat and tobacco were not significantly related to Escherichia coli 4.5S RNA, but were closely related to the 3'-terminus of bacterial 23S rRNA. Significant sequence homology was found between rat Novikoff hepatoma 4.5S RNAI and mouse and hamster 4.5S RNAs, suggesting that these RNAs are products of a family of genes with diverged sequences. E. coli 4.5S RNA had no significant sequence homology with any rodent 4.5S RNAs as a whole sequence. The E. coli, mouse and hamster 4.5S RNAs, however, were found to share a homologous 14-nucleotide sequence at the center of the molecules, which is known to exist as a conserved sequence in both Alu and Alu-equivalent sequences of mammalian DNAs.     

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.