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

Abstract

In some eukaryotes, a ‘hidden break’ has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.), but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although it has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and, in three of these, the break is located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28S rRNA and will be consistently underestimated in the great majority of animal species with a break.

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

“…One caveat of this measurement is that it is based on a Bayesian learning approach, and the samples used to train this algorithm were exclusively vertebrate [ 53 ]. As a result, the Bioanalyzer software sometimes fails to calculate RIN values of non-vertebrate samples as the peak shapes do not match training sets [ 53 , 54 ]. Moreover, a phenomenon known as the “hidden break” in the 28S ribosomal RNA causes it to break in two pieces of approximately the same size as the 18S [ 55 , 56 ], causing a sharp decrease in the RIN value of the sample that is not due to RNA degradation.…”

“…Moreover, a phenomenon known as the “hidden break” in the 28S ribosomal RNA causes it to break in two pieces of approximately the same size as the 18S [ 55 , 56 ], causing a sharp decrease in the RIN value of the sample that is not due to RNA degradation. This is triggered by denaturation, heating, or chemicals [ 55 , 56 ] and has been described to be widely present in platyhelminthes, including Schmidtea mediterranea , and many other animal groups [ 54 ]. As a result, RIN numbers of ACME samples are never above 8.…”

Lack of support for Deuterostomia prompts reinterpretation of the first Bilateria

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

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

“…The RNA integrity number (RIN) score was not able to be calculated due to the lack of 28S RNA-associated peak on electropherograms of L. stagnalis RNA. This phenomenon occurs due to a thermolabile hidden break in the molecule, as described in various invertebrates, which leads to its cleavage into two sub-parts of size similar to that of 18S RNA [ 100 ]. For this reason, RNA quality was evaluated using the global shape of migration profiles.…”