Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Gene Conversion amongst Alu SINE Elements

The process of non-allelic gene conversion acts on homologous sequences during recombination, replacing parts of one with the other to make them uniform. Such concerted evolution is best described as paralogous ribosomal RNA gene unification that serves to preserve the essential house-keeping functions of the converted genes. Transposed elements (TE), especially Alu short interspersed elements (SINE) that have more than a million copies in primate genomes, are a significant source of homologous units and a verified target of gene conversion. The consequences of such a recombination-based process are diverse, including multiplications of functional TE internal binding domains and, for evolutionists, confusing divergent annotations of orthologous transposable elements in related species. We systematically extracted and compared 68,097 Alu insertions in various primates looking for potential events of TE gene conversion and discovered 98 clear cases of Alu–Alu gene conversion, including 64 cases for which the direction of conversion was identified (e.g., AluS conversion to AluY). Gene conversion also does not necessarily affect the entire homologous sequence, and we detected 69 cases of partial gene conversion that resulted in virtual hybrids of two elements. Phylogenetic screening of gene-converted Alus revealed three clear hotspots of the process in the ancestors of Catarrhini, Hominoidea, and gibbons. In general, our systematic screening of orthologous primate loci for gene-converted TEs provides a new strategy and view of a post-integrative process that changes the identities of such elements.

Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3′ end of LINE-1_Cfs (i.e., LINE-1_Cf 3′-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.

Endogenous Double-Stranded RNA

The birth of long non-coding RNAs (lncRNAs) is closely associated with the presence and activation of repetitive elements in the genome. The transcription of endogenous retroviruses as well as long and short interspersed elements is not only essential for evolving lncRNAs but is also a significant source of double-stranded RNA (dsRNA). From an lncRNA-centric point of view, the latter is a minor source of bother in the context of the entire cell; however, dsRNA is an essential threat. A viral infection is associated with cytoplasmic dsRNA, and endogenous RNA hybrids only differ from viral dsRNA by the 5′ cap structure. Hence, a multi-layered defense network is in place to protect cells from viral infections but tolerates endogenous dsRNA structures. A first line of defense is established with compartmentalization; whereas endogenous dsRNA is found predominantly confined to the nucleus and the mitochondria, exogenous dsRNA reaches the cytoplasm. Here, various sensor proteins recognize features of dsRNA including the 5′ phosphate group of viral RNAs or hybrids with a particular length but not specific nucleotide sequences. The sensors trigger cellular stress pathways and innate immunity via interferon signaling but also induce apoptosis via caspase activation. Because of its central role in viral recognition and immune activation, dsRNA sensing is implicated in autoimmune diseases and used to treat cancer.

B2 SINE Copies Serve as a Transposable Boundary of DNA Methylation and Histone Modifications in the Mouse

More than one million copies of short interspersed elements (SINEs), a class of retrotransposons, are present in the mammalian genomes, particularly within gene-rich genomic regions. Evidence has accumulated that ancient SINE sequences have acquired new binding sites for transcription factors (TFs) through multiple mutations following retrotransposition, and as a result have rewired the host regulatory network during the course of evolution. However, it remains unclear whether currently active SINEs contribute to the expansion of TF binding sites. To study the mobility, expression, and function of SINE copies, we first identified about 2,000 insertional polymorphisms of SINE B1 and B2 families within Mus musculus. Using a novel RNA sequencing method designated as melRNA-seq, we detected the expression of SINEs in male germ cells at both the subfamily and genomic copy levels: the vast majority of B1 RNAs originated from evolutionarily young subfamilies, whereas B2 RNAs originated from both young and old subfamilies. DNA methylation and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses in liver revealed that polymorphic B2 insertions served as a boundary element inhibiting the expansion of DNA hypomethylated and histone hyperacetylated regions, and decreased the expression of neighboring genes. Moreover, genomic B2 copies were enriched at the boundary of various histone modifications, and chromatin insulator protein, CCCTC-binding factor, a well-known chromatin boundary protein, bound to >100 polymorphic and >10,000 non-polymorphic B2 insertions. These results suggest that the currently active B2 copies are mobile boundary elements that can modulate chromatin modifications and gene expression, and are likely involved in epigenomic and phenotypic diversification of the mouse species.

TATA-Like Boxes in RNA Polymerase III Promoters: Requirements for Nucleotide Sequences

tRNA and some other non-coding RNA genes are transcribed by RNA polymerase III (pol III), due to the presence of intragenic promoter, consisting of boxes A and B spaced by 30–40 bp. Such pol III promoters, called type 2, are also intrinsic to Short Interspersed Elements (SINEs). The contribution of 5′-flanking sequences to the transcription efficiency of genes containing type 2 promoters is still studied insufficiently. Here, we studied this issue, focusing on the genes of two small non-coding RNAs (4.5SH and 4.5SI), as well as B1 and B2 SINEs from the mouse genome. We found that the regions from position −31 to −24 may significantly influence the transcription of genes and SINEs. We studied the influence of nucleotide substitutions in these sites, representing TATA-like boxes, on transcription of 4.5SH and 4.5SI RNA genes. As a rule, the substitutions of A and T to G or C reduced the transcription level, although the replacement of C with A also lowered it. In 4.5SH gene, five distal nucleotides of −31/−24 box (TTCAAGTA) appeared to be the most important, while in the box −31/−24 of 4.5SI gene (CTACATGA), all nucleotides, except for the first one, contributed significantly to the transcription efficiency. Random sequences occurring at positions −31/−24 upstream of SINE copies integrated into genome, promoted their transcription with different efficacy. In the 5′-flanking sequences of 4.5SH and 4.5SI RNA genes, the recognition sites of CREB, C/EBP, and Sp1 factors were found, and their deletion decreased the transcription.

DNA Methylation Is Correlated with Gene Expression during Diapause Termination of Early Embryonic Development in the Silkworm (Bombyx mori)

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Riding the Wave: The SINE-Specific V Highly-Conserved Domain Spread into Mammalian Genomes Exploiting the Replication Burst of the MER6 DNA Transposon

Transposable elements are widely distributed within genomes where they may significantly impact their evolution and cell functions. Short interspersed elements (SINEs) are non-autonomous, fast-evolving elements, but some of them carry a highly conserved domain (HCD), whose sequence remained substantially unchanged throughout the metazoan evolution. SINEs carrying the HCD called V are absent in amniote genomes, but V-like sequences were found within the miniature inverted-repeat transposable element (MITE) MER6 in Homo sapiens. In the present work, the genomic distribution and evolution of MER6 are investigated, in order to reconstruct the origin of human V domain and to envisage its possible functional role. The analysis of 85 tetrapod genomes revealed that MER6 and its variant MER6A are found in primates, while only the MER6A variant was found in bats and eulipotyphlans. These MITEs appeared no longer active, in line with literature data on mammalian DNA transposons. Moreover, they appeared to have originated from a Mariner element found in turtles and from a V-SINE from bony fishes. MER6 insertions were found within genes and conserved in mRNAs: in line with previous hypothesis on functional role of HCDs, the MER6 V domain may be important for cell function also in mammals.

De novo genome and transcriptome analyses provide insights into the biology of the trematode human parasite Fasciolopsis buski

Many trematode parasites cause infection in humans and are thought to be a major public health problem. Their ecological diversity in different regions provides challenging questions on evolution of these organisms. In this report, we perform transcriptome analysis of the giant intestinal fluke, Fasciolopsis buski, using next generation sequencing technology. Short read sequences derived from polyA containing RNA of this organism were assembled into 30677 unigenes that led to the annotation of 12380 genes. Annotation of the assembled transcripts enabled insight into processes and pathways in the intestinal fluke, such as RNAi pathway and energy metabolism. The expressed kinome of the organism was characterized by identifying all protein kinases. We have also carried out whole genome sequencing and used the sequences to confirm absence of some of the genes, not observed in transcriptome data, such as genes involved in fatty acid biosynthetic pathway. Transcriptome data also helped us to identify some of the expressed transposable elements. Though many Long Interspersed elements (LINEs) were identified, only two Short Interspersed Elements (SINEs) were visible. Overall transcriptome and draft genome analysis of F. buski helped us to characterize some its important biological characteristics and provided enormous resources for development of a suitable diagnostic system and anti-parasitic therapeutic molecules.