Levels of sdRNAs in cytoplasm and their association with ribosomes are dependent upon stress conditions but independent from snoRNA expression

Anna M Mleczko,Piotr Machtel,Kamilla Bąkowska-Żywicka,Mateusz Walkowiak,Piotr J Pietras,Anna Wasilewska

doi:10.1038/s41598-019-54924-2

Anna M Mleczko, Piotr Machtel + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-54924-2

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Abstract

In recent years, a number of small RNA molecules derived from snoRNAs have been observed. Findings concerning the functions of snoRNA-derived small RNAs (sdRNAs) in cells are limited primarily to their involvement in microRNA pathways. However, similar molecules have been observed in Saccharomyces cerevisiae, which is an organism lacking miRNA machinery. Here we examined the subcellular localization of sdRNAs in yeast. Our findings reveal that both sdRNAs and their precursors, snoRNAs, are present in the cytoplasm at levels dependent upon stress conditions. Moreover, both sdRNAs and snoRNAs may interact with translating ribosomes in a stress-dependent manner. Likely consequential to their ribosome association and protein synthesis suppression features, yeast sdRNAs may exert inhibitory activity on translation. Observed levels of sdRNAs and snoRNAs in the cytoplasm and their apparent presence in the ribosomal fractions suggest independent regulation of these molecules by yet unknown factors.

Highlights

SnoRNAs are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosomal RNAs, respectively
These analyses demonstrated the ability of digital droplet PCR (ddPCR) to detect small RNA input levels, as low as 0.005 pg
We revealed that snoRNAs and snoRNA-derived small RNAs (sdRNAs) in Saccharomyces cerevisiae are present in the cytoplasm, where they associate with ribosomes[30]

Summary

Introduction

SnoRNAs are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosomal RNAs, respectively. Our recent work presented another possibility of sdRNA localization within the cytoplasm by association with ribosomes[8] These studies were performed in S. cerevisiae, an organism lacking miRNA machinery; one might suspect a distinct role for non-miRNA sdRNAs in yeast. A growing body of evidence has emerged linking both full-length snoRNAs and their derivatives to oncogenesis (reviewed in[21]) Considering both localization and the potential functions of full-length snoRNAs and sdRNAs, one might suspect that snoRNAs and their derivatives orchestrate responses to environmental stress outside of the nucleus. Yeast sdRNAs are observed in limited numbers during the sequencing of ribosome-associated small RNAs, with a maximum of 15 copies in the rancRNA library[8] Their regulatory potential cannot be excluded, as recent studies demonstrated that even relatively small levels (when compared to ∼200,000 ribosomes/cell) of ribosome-associated noncoding RNA (ranc_18mer, ∼27,000 molecules/cell) are sufficient to substantially influence global ribosome activity and regulate translation[22]. Poor sensitivity and low throughput of hybridization-based technologies can be overcome by sensitive, amplification-based detection methods, such as stem–loop reverse transcription PCR (SL-RT-PCR), originally described by Chen et al.[24] and successfully implemented by our group for detection of yeast sdRNAs from as little as 50 ng of low molecular weight cellular RNA23

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