Zooming in: PAGE-Northern Blot Helps to Analyze Anti-Sense Transcripts Originating from Human rIGS under Transcriptional Stress

Anastasia A Sadova,Dmitry Y Panteleev,Galina V Pavlova

doi:10.3390/ncrna7030050

Abstract

Ribosomal intergenic spacer (rIGS), located between the 45S rRNA coding arrays in humans, is a deep, unexplored source of small and long non-coding RNA molecules transcribed in certain conditions to help a cell generate a stress response, pass through a differentiation state or fine tune the functioning of the nucleolus as a ribosome biogenesis center of the cell. Many of the non-coding transcripts originating from the rIGS are not characterized to date. Here, we confirm the transcriptional activity of the region laying a 2 kb upstream of the rRNA promoter, and demonstrate its altered expression under transcriptional stress, induced by a wide range of known transcription inhibitors. We managed to show an increased variability of anti-sense transcripts in alpha-amanitin treated cells by applying the low-molecular RNA fraction extracted from agarose gel to PAGE-northern. Also, the fractioning of RNA by size using agarose gel slices occurred, being applicable for determining the sizes of target transcripts via RT-PCR.

Highlights

In the current work we investigate the phenomenon of the transcription in the prepromoter region of human ribosomal intergenic spacer, which lies between rRNAchromosomes
We were eager to know whether the anti-sense transcripts, which we have recently demonstrated to originate from the zone ~2000 bp upstream of the rDNA promoter [21]
We performed a northern blot analysis, which confirmed the increased amount of target transcripts, which presumably result from an enhanced transcriptional activity of the zone under investigation in alpha-amanitin treated cells compared to non-treated cells (Figure 2B)

Summary

Introduction

Much attention has been drawn to the investigation of small non-coding. RNA molecules, originating from different parts of the genome, which possess a myriad of diverse functions in living creatures [1,2]. Many researchers make use of contemporary approaches for the characterization of these transcripts such as bioinformatic analysis of various RNA-seq databases, and computer modelling based on already existing information on multiple classes of non-coding RNAs in different species [3,4]. This “dry-biology” data must be verified by in-lab experimental procedures.

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Discussion

Conclusion