Insects’ RNA Profiling Reveals Absence of “Hidden Break” in 28S Ribosomal RNA Molecule of Onion Thrips,Thrips tabaci

Rosaline Wanjiru Macharia,Erick Onyango Aroko,Fidelis Levi Ombura

doi:10.1155/2015/965294

Rosaline Wanjiru Macharia, Erick Onyango Aroko + Show 1 more

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https://doi.org/10.1155/2015/965294

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Abstract

With an exception of aphids, insects' 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect's RNA, the “hidden break” is absent in the 28S rRNA of onion thrips, Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies' 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect's 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

Highlights

Insects (Arthropoda: Insecta) constitute up to 55% of characterized species, playing different roles in the environment [1]
Despite many years of research on insect rRNA, misinterpretation of their RNA profiles is common to molecular biologists, partly due to the variations in 28S rRNA thermolability in different insect species
This dissociation of 28S rRNA molecule is attributed to the presence of an UAAU tract in their rRNA loop which acts as the cleavage site, known as the “hidden break” [6]

Summary

Introduction

Insects (Arthropoda: Insecta) constitute up to 55% of characterized species, playing different roles in the environment [1]. Isolation of high quality RNA from biological samples is challenged by the ubiquitous presence of ribonucleases (RNases) that rapidly degrade freshly prepared RNA [2] This makes the assessment of RNA quality mandatory prior to its downstream application. 28S rRNA from insects and most protostomes is characterized by its dissociation into two sized α and β subunits [5], which comigrates with the 18S rRNA to exhibit a single band profile. This dissociation of 28S rRNA molecule is attributed to the presence of an UAAU tract in their rRNA loop which acts as the cleavage site, known as the “hidden break” [6]. The pea aphid 28S rRNA, just like deuterostomes 28S rRNA, does not harbor the “hidden break” but instead

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