On the origin and evolution of RNA editing in metazoans

Extensive adenosine-to-inosine (A-to-I) editing of nuclear-transcribed mRNAs is the hallmark of metazoan transcriptional regulation. Here, by profiling the RNA editomes of 22 species that cover major groups of Holozoa, we provide substantial evidence supporting A-to-I mRNA editing as a regulatory innovation originating in the last common ancestor of extant metazoans. This ancient biochemistry process is preserved in most extant metazoan phyla and primarily targets endogenous double-stranded RNA (dsRNA) formed by evolutionarily young repeats. We also find intermolecular pairing of sense-antisense transcripts as an important mechanism for forming dsRNA substrates for A-to-I editing in some but not all lineages. Likewise, recoding editing is rarely shared across lineages but preferentially targets genes involved in neural and cytoskeleton systems in bilaterians. We conclude that metazoan A-to-I editing might first emerge as a safeguard mechanism against repeat-derived dsRNA and was later co-opted into diverse biological processes due to its mutagenic nature

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Repetitive element annotation for the eastern banjo frog genome assembly v2

Repetitive elements in the Limnodynastes dumerilii dumerilii genome assembly were identified by RepeatMasker, RepeatProteinMask, RepeatModeler and Tandem Repeats Finder. </p

Repetitive element annotation for the eastern banjo frog genome assembly v3

Repetitive elements in the Limnodynastes dumerilii dumerilii genome assembly were identified by RepeatMasker, RepeatProteinMask, RepeatModeler and Tandem Repeats Finder. </p

Engineering the Active Site Pocket to Enhance the Catalytic Efficiency of a Novel Feruloyl Esterase Derived From Human Intestinal Bacteria Dorea formicigenerans

The human gut microbiota play essential roles in metabolism and human health, especially by enzymatically utilizing dietary fiber that the host cannot directly digest and releasing functional components including short-chain fatty acids (SCFAs) and hydroxycinnamic acids (e.g., ferulic acid). In our previous study, seven potential feruloyl esterase (FAE) genes were identified from the gut microbiota. In the current work, one of the genes encoding a novel FAE (DfFAE) from Dorea formicigenerans of Firmicutes was bacterially expressed, purified and characterized. The 30.5 kDa type-A DfFAE has an optimum pH and temperature of 8.4 and 40 °C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five α-helices cap domain that shaped an unusually narrow and deep active site pocket containing a specific substrate access tunnel in DfFAE. Furthermore, rational design strategy was subjected to the active site pocket in an aim of improving its enzymatic activities. The mutants V252A, N156A, W255A, P149A, and P186A showed 1.8 to 5.7-fold increase in catalytic efficiency toward pNPA, while W255A also exhibited altered substrate preference toward long-chain substrate pNPO (45.5-fold). This study highlighted an unusual active site architecture in DfFAE that influenced its substrate selectivity and illustrated the applicability of rational design for enhanced enzymatic properties.</jats:p

A draft genome assembly of the eastern banjo frog Limnodynastes dumerilii dumerilii (Anura: Limnodynastidae)

Amphibian genomes are usually challenging to assemble due to their large genome size and high repeat content. The Limnodynastidae is a family of frogs native to Australia, Tasmania and New Guinea. As an anuran lineage that successfully diversified on the Australian continent, it represents an important lineage in the amphibian tree of life but lacks reference genomes. Here we sequenced and annotated the genome of the eastern banjo frog Limnodynastes dumerilii dumerilii to fill this gap. The total length of the genome assembly is 2.38 Gb with a scaffold N50 of 285.9 kb. We identified 1.21 Gb of non-redundant sequences as repetitive elements and annotated 24,548 protein-coding genes in the assembly. BUSCO assessment indicated that more than 94% of the expected vertebrate genes were present in the genome assembly and the gene set. We anticipate that this annotated genome assembly will advance the future study of anuran phylogeny and amphibian genome evolution. </jats:p

Quality control protocol for the raw sequencing reads of the eastern banjo frog v1

The raw sequencing data from the 14 libraries (170 bp × 1, 250 bp × 1, 500 bp × 1, 800 bp × 1, 2 kb × 3, 5 kb × 3, 10 kb × 2, and 20 kb × 2) were subjected to strict quality control by SOAPnuke (v1.5.3) prior to downstream analyses. Briefly, for the raw reads from each library, we trimmed the unreliable bases at the head and tail of each read where the per-position GC content was unbalanced or the per-position base quality was low across all reads; we removed the read pairs with adapter contamination, with high proportion of low-quality or unknown (N) bases; we removed duplicate read pairs resulted from polymerase chain reaction (PCR) amplification (i.e. PCR duplicates); and we also removed the overlapping read pairs in all but the 170 bp and 250 bp libraries where the paired reads were expected to be overlapping. </p

Protein-coding gene annotation for the eastern banjo frog genome assembly v2

We first used homology-based and de novo predictions to build gene models for the Limnodynastes dumerilii dumerilii genome assembly. Then, we searched theLimnodynastes dumerilii dumerilii proteins against public databases to perform functional annotation. </p

A draft genome assembly of the eastern banjo frog <i>Limnodynastes dumerilii dumerilii</i> (Anura: Limnodynastidae)

AbstractAmphibian genomes are usually challenging to assemble due to large genome size and high repeat content. The Limnodynastidae is a family of frogs native to Australia, Tasmania and New Guinea. As an anuran lineage that successfully diversified on the Australian continent, it represents an important lineage in the amphibian tree of life but lacks reference genomes. Here we sequenced and annotated the genome of the eastern banjo frog Limnodynastes dumerilii dumerilii to fill this gap. The total length of the genome assembly is 2.38 Gb with a scaffold N50 of 285.9 kb. We identified 1.21 Gb of non-redundant sequences as repetitive elements and annotated 24,548 protein-coding genes in the assembly. BUSCO assessment indicated that more than 94% of the expected vertebrate genes were present in the genome assembly and the gene set. We anticipate that this annotated genome assembly will advance the future study of anuran phylogeny and amphibian genome evolution.</jats:p

Protein-coding gene annotation for the eastern banjo frog genome assembly v3

We first used homology-based and de novo predictions to build gene models for the Limnodynastes dumerilii dumerilii genome assembly. Then, we searched theLimnodynastes dumerilii dumerilii proteins against public databases to perform functional annotation. </p