A rapid and sensitive non‐radioactive method applicable for genome‐wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

Abstract

The conventional small RNA isolation and detection methods for yeast cells have been designed for a small number of samples. In order to conduct a genome-wide assessment of how each gene product impacts upon small non-coding RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive nonradioactive Northern method for RNA detection. The RNA isolation procedure is highly reproducible and requires only 4 hours for processing 96 samples, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-level small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analysis of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe, and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking, and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots.

Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

“…Nuclear and cytoplasmic RNA was extracted according to manufacturer’s directions using TRIzol-LS (Thermo Fisher) added directly to the final lysates. Northern blotting protocols were adapted from published sources (Rio, 2014; Wu et al, 2013). Following purification of RNA, nuclear and cytoplasmic cell equivalents from 500,000 (for Sybr Gold staining) or 50,000 (for Northern blots) cells were loaded on a 10% acrylamide-urea/TBE gel.…”

Sharing the load: Mex67–Mtr2 cofunctions with Los1 in primary tRNA nuclear export

“…Two micrograms of total RNA was separated by electrophoresis on urea-PAGE and transferred onto a Hybond N + membrane (Amersham), and specific tRNAs were detected by probing the membranes with digoxigenin-labeled probes as indicated in the figures and described previously (Wu et al 2013). Equal loading of total small RNA in gels was verified by staining the gels using SYBR Gold nucleic acid gel stain (ThermoFisher) or ethidium bromide (as indicated in figures).…”

RNA editing, epitranscriptomics, and processing in cancer progression

“…Following up on one intriguing phenotype from the screen, Dr. Hopper and her student, J. Wu, discovered that intron degradation in yeast requires that the intron first be phosphorylated by the enzyme with both tRNA ligase and kinase activities that ligates exons after splicing. 24 Notably, knocking down the human kinase Clp1, also involved in splicing tRNA introns, leads to the accumulation of free introns in human cells. Point mutations in this gene have been found in patients with neurological disorders, raising the intriguing question of whether aberrant accumulation of tRNA introns could be responsible for neurological phenotypes.…”