Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

Fowler, Emily K., Mohorianu, Irina, Smith, Damian T., Dalmay, Tamas ORCID: https://orcid.org/0000-0003-1492-5429 and Chapman, Tracey (2018) Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues. PLoS One, 13 (2). ISSN 1932-6203

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Abstract

RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues.

Item Type:	Article
Uncontrolled Keywords:	small rna (srna),microrna (mirna),rna-seq,blocking oligo(s),accessory glands,fruit flies,drosophila melanogaster
Faculty \ School:	Faculty of Science > School of Biological Sciences Faculty of Science > School of Computing Sciences
UEA Research Groups:	Faculty of Science > Research Centres > Centre for Ecology, Evolution and Conservation Faculty of Science > Research Groups > Organisms and the Environment Faculty of Science > Research Groups > Plant Sciences
Depositing User:	Pure Connector
Date Deposited:	02 Feb 2018 10:30
Last Modified:	13 May 2023 00:26
URI:	https://ueaeprints.uea.ac.uk/id/eprint/66187
DOI:	10.1371/journal.pone.0191966

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