Comparative Mitogenomics of Two Critically Endangered Turtles, Batagur Kachuga and Batagur Dhongoka (Testudines: Geoemydidae): Implications in Phylogenetics of Freshwater Turtles

The Red-crowned roofed turtle (Batagur kachuga) and Three-striped roofed turtle (B. dhongoka) are ‘critically endangered’ turtles in the Geoemydidae family. Herein, we generated the novel mitochondrial genome sequence of B. kachuga (16,155) and B. dhongoka (15,620) and compared it with other turtles species. Batagur mitogenome has 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), and one control region (CR). The genome composition was biased toward A + T, with positive AT-skew and negative GC-skew. In the examined species, all 13 PCGs were started by ATG codons, except COI gene, which was initiated by GTG. The majority of mito-genes were encoded on the heavy strand, except eight tRNAs and the ND6 region. We observed a typical cloverleaf structure for all tRNA, excluding tRNASer (AGN), where the base pairs of the dihydrouridine (DHU) arm were abridged. Bayesian Inference (BI) based phylogenetic analysis was constructed among 39 species from six Testudines families, exhibited a close genetic relationship between Batagur and Pangshura with a high supporting value (PP ~ 0.99). It provides additional mitogenomic resources for further study of the Testudines evolutionary patterns.

Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

Background The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive. Results Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3′-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

The Complete Mitochondrial Genome of endemic giant tarantula, Lyrognathus crotalus (Araneae: Theraphosidae) and comparative analysis

The complete mitochondrial genome of Lyrognathus crotalus is sequenced, annotated and compared with other spider mitogenomes. It is 13,865 bp long and featured by 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and a control region (CR). Most of the PCGs used ATN start codon except cox3, and nad4 with TTG. Comparative studies indicated the use of TTG, TTA, TTT, GTG, CTG, CTA as start codons by few PCGs. Most of the tRNAs were truncated and do not fold into the typical cloverleaf structure. Further, the motif (CATATA) was detected in CR of nine species including L. crotalus. The gene arrangement of L. crotalus compared with ancestral arthropod showed the transposition of five tRNAs and one tandem duplication random loss (TDRL) event. Five plesiomophic gene blocks (A-E) were identified, of which, four (A, B, D, E) retained in all taxa except family Salticidae. However, block C was retained in Mygalomorphae and two families of Araneomorphae (Hypochilidae and Pholcidae). Out of 146 derived gene boundaries in all taxa, 15 synapomorphic gene boundaries were identified. TreeREx analysis also revealed the transposition of trnI, which makes three derived boundaries and congruent with the result of the gene boundary mapping. Maximum likelihood and Bayesian inference showed similar topologies and congruent with morphology, and previously reported multi-gene phylogeny. However, the Gene-Order based phylogeny showed sister relationship of L. crotalus with two Araneomorphae family members (Hypochilidae and Pholcidae) and other Mygalomorphae species.

Mitochondrial genome of the sweet potato hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), and comparison with other Lepidoptera species

In the present study, we sequenced the complete mitochondrial genome (mitogenome) of Agrius convolvuli (Lepidoptera: Sphingidae) and compared it with previously sequenced mitogenomes of lepidopteran species. The mitogenome was a circular molecule, 15 349 base pairs (bp) long, containing 37 genes. The order and orientation of genes in the A. convolvuli mitogenome were similar to those in sequenced mitogenomes of other lepidopterans. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which seemed to be initiated by the codon CGA, as observed in other lepidopterans. Three of the 13 PCGs had the incomplete termination codon T, while the remainder terminated with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Asn, Ile, Leu2, Lys, Phe, and Tyr were the most frequently used codon families. All transfer RNAs were folded into the expected cloverleaf structure except for tRNASer(AGN), which lacked a stable dihydrouridine arm. The length of the adenine (A) + thymine (T)-rich region was 331 bp. This region included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA)8 element next to the motif ATTTA. Phylogenetic analyses (maximum likelihood and Bayesian methods) showed that A. convolvuli belongs to the family Sphingidae.

Comparative Mitochondrial Genome Analysis of Eligma narcissus and other Lepidopteran Insects Reveals Conserved Mitochondrial Genome Organization and Phylogenetic Relationships

In this study, we sequenced the complete mitochondrial genome of Eligma narcissus and compared it with 18 other lepidopteran species. The mitochondrial genome (mitogenome) was a circular molecule of 15,376 bp containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and an adenine (A) + thymine (T) − rich region. The positive AT skew (0.007) indicated the occurrence of more As than Ts. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. All PCGs were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by the CGA sequence, as observed in other lepidopterans. The results of the codon usage analysis indicated that Asn, Ile, Leu, Tyr and Phe were the five most frequent amino acids. All tRNA genes were shown to be folded into the expected typical cloverleaf structure observed for mitochondrial tRNA genes. Phylogenetic relationships were analyzed based on the nucleotide sequences of 13 PCGs from other insect mitogenomes, which confirmed that E. narcissus is a member of the Noctuidae superfamily.

Shaping tRNA

This model-building activity provides a quick, visual, hands-on tool that allows students to examine more carefully the cloverleaf structure of a typical tRNA molecule. When used as a supplement to lessons that involve gene expression, this exercise reinforces several concepts in molecular genetics, including nucleotide base-pairing rules, the importance of molecular shape, the consequences of genetic mutation, and similarities and differences between DNA and RNA.

The mitochondrial genome of Coridius chinensis (Hemiptera: Dinidoridae)

The nearly complete mitochondrial genome of Coridius chinensis (Dallas) is reported in this study. The mitogenome is a double-stranded circular molecule of more than 14,648 bp in length with an A+T content of 75.1%. It encoded 37 genes as in other insect mtDNAs, including 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and a control region (unsuccessful sequencing), and the gene order is the same as most other known heteropteran mitogenomes. All of the 22 transfer RNAs can be folded into the typical cloverleaf structure except tRNASer(AGN), which can only form a simple loop at the site of dihydrouridine (DHU) arm as known in other metazoans. The secondary structures of the large and small ribosomal RNAs of C. chinensis are similar to other presented insects. The rrnL consisted of six structural domains and 40 helices, and the rrnS consisted of three structural domains and 26 helices. Nine PCGs are initiated with the standard initiation codons (ATN), while ND6 and ND1 use GTG, and COI and ATP8 use TTG. All PCGs stopped with TAA/TAG termination codons except the COII terminated with a single T residue. Asymmetry in the nucleotide composition between J-strand and N-strand was observed in this mitogenome.

Sequencing of Porcine Enterovirus Groups II and III Reveals Unique Features of Both Virus Groups

ABSTRACT The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5′-cloverleaf structure.