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.     

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.     

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.     

SINEs

SINEs with internal promoters for RNA polymerase III are ubiquitous in the genomes of the animal kingdom, including invertebrates. Although the human Alu family, and related families, originates from 7SL RNA, all other SINEs originate from tRNA. SINEs have been amplified many times, altered in genomic organization and fixed in the population at certain stages of evolution. They can therefore be regarded as time-landmarks of evolution. It is proposed that both population genetics and molecular biology are required for understanding the expansion of SINEs.     

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.     

Analysis of DNA of higher primates using inter-SINE PCR

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.     

Analysis of DNA of higher primates using inter-SINE PCR

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.     

Alu insertion loci and platyrrhine primate phylogeny

Short INterspersed Elements (SINEs) make very useful phylogenetic markers because the integration of a particular element at a location in the genome is irreversible and of known polarity. These attributes make analysis of SINEs as phylogenetic characters an essentially homoplasy-free affair. Alu elements are primate-specific SINEs that make up a large portion of the human genome and are also widespread in other primates. Using a combination wet-bench and computational approach we recovered 190 Alu insertions, 183 of which are specific to the genomes of nine New World primates. We used these loci to investigate branching order and have produced a cladogram that supports a sister relationship between Atelidae (spider, woolly, and howler monkeys) and Cebidae (marmosets, tamarins, and owl monkeys) and then the joining of this two family clade to Pitheciidae (titi and saki monkeys). The data support these relationships with a homoplasy index of 0.00. In this study, we report one of the largest applications of SINE elements to phylogenetic analysis to date, and the results provide a robust molecular phylogeny for platyrrhine primates.     

Mosaic evolution of rodent B1 elements

We have determined sequences of PCR-amplified B 1 elements from hamster and rat (Myomorpha), chipmunk (Sciuromorpha), and guinea pig (Caviomorpha). Between three and six B 1 subfamilies were found in these species. In the phylogenetic analysis Bl sequences of hamster, mouse, and rat clustered separately from those of chipmunk and those of guinea pig. This is consistent with an independent evolution of B1 elements in separate rodent lineages. We exclude the possibility of convergent mutations to explain certain diagnostic characters within the modern B1 quasi-dimers and view these elements as mosaic structures assembling preexisting mutations. Furthermore, the presence of Alu-like structural motifs supports the hypothesis of the monophyletic origin of Alu and B1 repeats, i.e., from a common 7SL RNA-derived retroposing monomeric element. Correspondence to: E. Zitkiewicz     

Sequence composition similarities with the 7SL RNA are highly predictive of functional genomic features

Transposable elements derived from the 7SL RNA gene, such as Alu elements in primates, have had remarkable success in several mammalian lineages. The results presented here show a broad spectrum of functions for genomic segments that display sequence composition similarities with the 7SL RNA gene. Using thoroughly documented loci, we report that DNaseI-hypersensitive sites can be singled out in large genomic sequences by an assessment of sequence composition similarities with the 7SL RNA gene. We apply a root word frequency approach to illustrate a distinctive relationship between the sequence of the 7SL RNA gene and several classes of functional genomic features that are not presumed to be of transposable origin. Transposable elements that show noticeable similarities with the 7SL sequence include Alu sequences, as expected, but also long terminal repeats and the 5′-untranslated regions of long interspersed repetitive elements. In sequences masked for repeated elements, we find, when using the 7SL RNA gene as query sequence, distinctive similarities with promoters, exons and distal gene regulatory regions. The latter being the most notoriously difficult to detect, this approach may be useful for finding genomic segments that have regulatory functions and that may have escaped detection by existing methods.     

A mobile element based phylogeny of Old World monkeys

SINEs (Short INterspersed Elements) are a class of non-autonomous mobile elements that are <500 bp in length and have no open reading frames. Individual SINE elements are essentially homoplasy free with known ancestral states, making them useful genetic systems for phylogenetic studies. Alu elements are the most successful SINE in primate genomes and have been utilized for resolving primate phylogenetic relationships and human population genetics. However, no Alu based phylogenetic analysis has yet been performed to resolve relationships among Old World monkeys. Using both a computational approach and polymerase chain reaction display methodology, we identified 285 new Alu insertions from sixteen Old World monkey taxa that were informative at various levels of catarrhine phylogeny. We have utilized these elements along with 12 previously reported loci to construct a phylogenetic tree of the selected taxa. Relationships among all major clades are in general agreement with other molecular and morphological data sets but have stronger statistical support.     

A SINE in the genome of the cephalochordate amphioxus is an Alu element

Transposable elements of about 300 bp, termed "short interspersed nucleotide elements or SINEs are common in eukaryotes. However, Alu elements, SINEs containing restriction sites for the AluI enzyme, have been known only from primates. Here I report the first SINE found in the genome of the cephalochordate, amphioxus. It is an Alu element of 375 bp that does not share substantial identity with any genomic sequences in vertebrates. It was identified because it was located in the FoxD regulatory region in a cosmid derived from one individual, but absent from the two FoxD alleles of BACs from a second individual. However, searches of sequences of BACs and genomic traces from this second individual gave an estimate of 50-100 copies in the amphioxus genome. The finding of an Alu element in amphioxus raises the question of whether Alu elements in amphioxus and primates arose by convergent evolution or by inheritance from a common ancestor. Genome-wide analyses of transposable elements in amphioxus and other chordates such as tunicates, agnathans and cartilaginous fishes could well provide the answer.     

Most human Alu and murine B1 repeats are unique

Alus and B1s are short interspersed repeat elements (SINEs) indirectly derived from the 7SL RNA gene. While most researchers recognize that there exists extensive variability between individual elements, the extent of this variability has never been systematically tested. We examined all Alu elements over 200 nucleotides and all B1 elements over 100 nucleotides in the human and mouse genomes, and analyzed the number of copies of each element at various stringencies from 22 nucleotides to full length. Over 98% of 923,277 Alus and 365,377 B1s examined were unique when queried at full length. When the criterion was reduced to half the length of the repeat, 97% of the Alus and 73% of the B1s were still found to be a single copy. All single and multi-copy sequences have been mapped and documented. Access to the data is possible using the AluPlus website http://www.ibr.hawaii.edu.     

An alu-based phylogeny of lemurs (infraorder: lemuriformes)

LEMURS (INFRAORDER: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ～55-60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of Eulemur and Varecia, with Varecia the sister lineage to the other three genera.     

cis-Acting determinants of 7SL RNA packaging by HIV-1

The host noncoding RNA 7SL is highly enriched in the virions of retroviruses. We examined the regions of 7SL that mediate packaging by HIV-1. Both the Alu domain and the S domain were sufficient to mediate specific packaging when expressed separately as truncations of 7SL. However, while the Alu domain competed with endogenous 7SL for packaging in proportion to Gag, the S domain was packaged additively, implying that the Alu and S domains are packaged via separate mechanisms and that the Alu domain is packaged by the same mechanism as endogenous 7SL. Further truncations of the Alu domain or mutation of the Alu domain helix 5c region significantly reduced packaging efficiency, implicating helix 5c as critical for packaging, reinforcing the finding that 7SL packaging is highly selective, and confirming that 7SL is not passively acquired. Surprisingly, when the Alu domain was mutated so that it no longer contained a binding site for the SRP protein heterodimer SRP9/14, it was no longer packaged in a competitive manner but instead was packaged additively with endogenous 7SL. These data support a model in which 7SL RNA is packaged via interactions between Gag and a 7SL RNA structure that exists transiently at a discrete stage of SRP biogenesis. Our data further indicate that a secondary "additive" pathway exists that can result in the packaging of certain 7SL derivatives in molar excess to endogenously packaged 7SL.