Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution

NAT10 is an essential enzyme that catalyzes the formation of N4-acetylcytidine (ac4C) in eukaryotic transfer RNA (tRNA) and 18S ribosomal RNA (rRNA). Recent studies in human cells suggested that rRNA acetylation is dependent on SNORD13, a non-canonical box C/D small nucleolar RNA (SNORD) predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10 essential function in pre-rRNA processing remain to be defined. Here, we used CRISPR-Cas9 genome editing to formally demonstrate that SNORD13 is required for acetylation of a single cytidine residue of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for yeast or human cell growth, ribosome biogenesis, translation, and the development of multicellular metazoan model organisms. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation machinery that led to the characterization of many novel SNORD13 genes in phylogenetically-distant metazoans and more deeply rooted photosynthetic organisms. This includes an atypical SNORD13-like RNA in D. melanogaster which appears to guide ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that C. elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Altogether, our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across the eukaryotic tree of life and raise new questions regarding the biological function and evolutionary persistence of this highly conserved rRNA base modification.

hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA.

5-Methylcytosine (m5C) is a base modification broadly found on a variety of RNAs in the human transcriptome. In eukaryotes m5C is catalyzed by enzymes of the NSUN family, which is composed of seven members in humans (NSUN1-7). NOP2/NSUN1 has been mostly characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S rRNA. Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions remain poorly characterized. To define the roles of human NOP2/NSUN1, we used an miCLIP-seq approach to identify its RNA substrates. Our analysis reveals that vault RNA 1.2 and rRNA are NOP2/NSUN1-specific methylated targets and we further confirm by bisulfite sequencing that NOP2/NSUN1 is responsible for the deposition of m5C at residue 4447 on the 28S rRNA. Depletion of NOP2/NSUN1 impairs cell proliferation, rRNA processing and 60S ribosome biogenesis. Additionally, we find that NOP2/NSUN1 binds to the 5′ETS region of the pre-rRNA transcript and regulates pre-rRNA processing in part through non-catalytic complex formation with box C/D snoRNAs. Our study identifies for the first time the RNA substrates of human NOP2/NSUN1 and reveals additional functions in rRNA processing beyond catalyzing m5C base modification.

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA

In eukaryotes, 5-methylcytosine is the predominant DNA base modification, followed by N6-methyladenine. However, N4-methylcytosine (4mC) is confined to bacteria. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, freshwater invertebrates with transposon-poor genomes that are rich in foreign genes, lack C5-methyltransferases but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT introduces 4mC into DNA, and its chromodomain shapes the "histone-read-DNA-write" architecture together with a "DNA-read-histone-write" SETDB1/eggless H3K9me3 histone methyltransferase variant preferentially binding 4mC-DNA, to maintain 4mC and silent chromatin at transposons and tandem repeats. Our results bring the third base modification into the eukaryotic repertoire, demonstrate how non-native DNA methyl groups can reshape complex epigenetic systems to suppress transposon proliferation, and establish horizontal gene transfer as the source of regulatory innovation in eukaryotes.

Complete Genome Sequence and Methylome of the Type Strain of Shewanella algae

We report the complete genome sequence and base modification analysis of the Shewanella algae type strain CECT 5071 (= OK-1 = ATCC 51192 = DSM 9167 = IAM 14159). The genome is composed of a single chromosome of 4,924,764 bp, with a GC content of 53.10%.

m6A is required for resolving progenitor identity during planarian stem cell differentiation

Regeneration requires accurate production of missing cell lineages. Cell production is driven by changes to gene expression, which is shaped by multiple layers of regulation. Here, we find that the ubiquitous mRNA base-modification, m6A, is required for proper cell fate choice and cellular maturation in planarian stem cells (neoblasts). We mapped m6A-enriched regions in 7,600 planarian genes, and found that perturbation of the m6A pathway resulted in progressive deterioration of tissues and death. Using single cell RNA sequencing of >20,000 cells following perturbation of the pathway, we discovered that m6A negatively regulates transcription of histone variants, and that inhibition of the pathway resulted in accumulation of undifferentiated cells throughout the animal in an abnormal transcriptional state. Analysis of >1000 planarian gene expression datasets revealed that the inhibition of the chromatin modifying complex NuRD had almost indistinguishable consequences, unraveling an unappreciated link between m6A and chromatin modifications. Our findings reveal that m6A is critical for planarian stem cell homeostasis and gene regulation in regeneration.

Hopeful monsters: unintended sequencing of famously malformed mite mitochondrial tRNAs reveals widespread expression and processing of sense–antisense pairs

Although tRNA structure is one of the most conserved and recognizable shapes in molecular biology, aberrant tRNAs are frequently found in the mitochondrial genomes of metazoans. The extremely degenerate structures of several mitochondrial tRNAs (mt-tRNAs) have led to doubts about their expression and function. Mites from the arachnid superorder Acariformes are predicted to have some of the shortest mt-tRNAs, with a complete loss of cloverleaf-like shape. While performing mitochondrial isolations and recently developed tRNA-seq methods in plant tissue, we inadvertently sequenced the mt-tRNAs from a common plant pest, the acariform mite Tetranychus urticae, to a high enough coverage to detect all previously annotated T. urticae tRNA regions. The results not only confirm expression, CCA-tailing and post-transcriptional base modification of these highly divergent tRNAs, but also revealed paired sense and antisense expression of multiple T. urticae mt-tRNAs. Mirrored expression of mt-tRNA genes has been hypothesized but not previously demonstrated to be common in any system. We discuss the functional roles that these divergent tRNAs could have as both decoding molecules in translation and processing signals in transcript maturation pathways, as well as how sense–antisense pairs add another dimension to the bizarre tRNA biology of mitochondrial genomes.

Lost in the Crowd: How Does Human 8-Oxoguanine DNA Glycosylase 1 (OGG1) Find 8-Oxoguanine in the Genome?

The most frequent DNA lesion resulting from an oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a premutagenic base modification due to its capacity to pair with adenine. Thus, the repair of 8-oxoG is critical for the preservation of the genetic information. Nowadays, 8-oxoG is also considered as an oxidative stress-sensor with a putative role in transcription regulation. In mammalian cells, the modified base is excised by the 8-oxoguanine DNA glycosylase (OGG1), initiating the base excision repair (BER) pathway. OGG1 confronts the massive challenge that is finding rare occurrences of 8-oxoG among a million-fold excess of normal guanines. Here, we review the current knowledge on the search and discrimination mechanisms employed by OGG1 to find its substrate in the genome. While there is considerable data from in vitro experiments, much less is known on how OGG1 is recruited to chromatin and scans the genome within the cellular nucleus. Based on what is known of the strategies used by proteins searching for rare genomic targets, we discuss the possible scenarios allowing the efficient detection of 8-oxoG by OGG1.