Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers

Abstract

10In some eukaryotes, a 'hidden break' has been described in which the 28S ribosomal RNA molecule is 11 cleaved into two subparts. The break is common in protostome animals but has also been reported in 12 some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine 13 the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find an homologous 14 break is present across protostomes although has been lost in some taxa. We show that rare breaks in 15 vertebrates are not homologous to the protostome break. A break is found in just 3 out of 307 species of 16 non-animal eukaryotes studied but these are located in the same position as the protostome break 17 suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28s 18 rRNA and will be consistently underestimated in the great majority of animal species with a break. 193 Introduction. 20Ribosomes are made up of up to 80 ribosomal proteins and three (in prokaryotes) or four (in eukaryotes) 21 structural ribosomal RNAs named according to their sizes: in eukaryotes these are the 5S (~120 22 nucleotides), the 5.8S (~150 nucleotides), the 18S (~1800 nucleotides) and the 28S (~4000 to 5000 23 nucleotides). The 5.8S, 18S and 28S rRNAs are initially transcribed as a single RNA operon (the 5S is at a 24 separate locus in eukaryotes). The 18S and 5.8S are separated by the Internal Transcribed Spacer 1 (ITS1) 25 and 5.8S and 28S are separated by ITS2. The initial transcript is cleaved into three functional RNAs by 26 removing ITS1 and ITS2 (Fig. 1A). 28In some species this picture is complicated by observations of a 'hidden break' in the 28S ribosomal RNA. 29In organisms with a hidden break, the 28S rRNA molecule itself is cleaved into two approximately equal 30 sized molecules of ~2000 nucleotides each (Fig. 1A) (Winnebeck et al., 2010). These two RNAs (the 5' 31 28Sa and the 3' 28Sb) are nevertheless intimately linked by inter-molecular hydrogen bonding within the 32 large subunit of the ribosome, just as the 28S and 5.8S rRNAs as well as different regions of the intact 33 28S rRNA are in species without a break. 35The hidden break, first described in pupae of the silkmoth Hyalophora cecropia (Applebaum et al., 1966) 36 manifests itself experimentally when total RNA is extracted and separated according to size using gel 37 electrophoresis. If the RNA is first denatured by heating in order to separate hydrogen bonded stretches 38 of RNAs before electrophoresis, the hydrogen bonded 28Sa and 28Sb subunits separate. These two 39 molecules, being approximately the same size, then migrate on a gel at the same rate as each other and, 40 coincidentally, at the same rate as the almost identically sized 18S rRNA molecule. The effect is that, rather 41 than observing two distinct ribosomal RNA bands on the gel of ~2000 and ~4000 nucleotides, a single 42 (and more intense) band composed of three different molecules each approximately 2kb long (18S, 43 28Sa and 28Sb) is seen (McCarthy et ...

Lack of support for Deuterostomia prompts reinterpretation of the first Bilateria

“…Protostomes have 12 unique microRNA families 13 compared to 1 in deuterostomes and protostomes share 58 'near intron pairs' compared to just 7 in deuterostomes. Protostomes also share a highly distinct, conserved variant of the mitochondrial NAD5 protein 14 and a hidden break in their 28S ribosomal RNA 15 .…”

Shedding light: a phylotranscriptomic perspective illuminates the origin of photosymbiosis in marine bivalves

“…mRNA quality was assessed with a picoRNA assay in an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantity was measured in a Qubit fluorometer (Life Technologies). 28S rRNA in mollusk samples breaks down into two segments of comparable sizes to 18S rRNA during Bioanalyser quality assessment, resulting in non-meaningful RIN scores [44][45][46]. Therefore, we used the RNA electropherogram itself to validate the RNA integrity.…”

Shedding light: A phylotranscriptomic perspective illuminates the origin of photosymbiosis in marine bivalves

“…mRNA quality was assessed with a picoRNA assay in an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantity was measured in a Qubit fluorometer (Life Technologies). 28S rRNA in mollusk samples breaks down into two segments of comparable sizes to 18S rRNA during Bioanalyser quality assessment, resulting in non-meaningful RIN scores [44][45][46].…”