In-Gel Digestion for Mass Spectrometric Characterization of RNA from Fluorescently Stained Polyacrylamide Gels

Abstract

Although current mass spectrometry-based proteomics technology allows for high-throughput analysis of protein components in functional ribonucleoprotein complexes, this technology has had limited application to studies of RNA itself. Here we present a protocol for RNA analysis using polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Specifically, RNAs of interest are subjected to polyacrylamide gel electrophoresis and stained with a fluorescent dye, and RNAs in gel bands are digested with nuclease and then analyzed directly liquid chromatography-mass spectrometry, resulting in highly accurate mass values and reliable information on post-transcriptional modifications. We demonstrate that the method can be applied to the identification and chemical analysis of small RNAs in mouse embryonic stem cell extracts and of small RNAs in the spliceosomal ribonucleoprotein complex pulled down from yeast cells using a tagged protein cofactor as bait. The protocol is relatively simple and allowed us to identify not only three novel methylated nucleotide residues of RNase P RNA, U6 snRNA, and 7SL RNA prepared from mouse ES cells but also various 3'-end forms of U4, U5S, and U6 snRNAs isolated from the yeast spliceosome at the femtomole level. The method is thus a convenient tool for direct analysis of RNAs in various cellular ribonucleoprotein complexes, particularly for the analysis of post-transcriptional modifications and metabolic processing of RNA.

Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis

“…The product ions included the major c/y and a/w series with minor derivatives (dehydrated ions and ions that lost nucleotide bases; Supplementary Figure S2A and Supplementary Tables S1 and S2), as reported (33). Among the oligonucleotides generated from the U1 snRNA fractions (Figure 1C), we mainly detected m 3 GpppAmUmACUUACCUGp (or m 3 GpppAmUmACΨΨACCUGp) (containing a 5′ cap of 2,2,7-trimethylguanosine, m 3 G) that originated from the 5′ end (Figures 2A, Supplementary Figure S2B and C and Supplementary Table S2) and CUUUCCCCUG-OH from the 3′ end (Supplementary Figure S2D and Supplementary Table S2), indicating that the oligonucleotides mostly originated from mature U1 snRNA.…”

“…RNAs were detected by monitoring A 260 and, where necessary, the fractionated RNA was digested with RNase T1 (10 ng) in 10 mM sodium acetate (pH 5.3) at 37°C for 60 min. The resulting oligonucleotide mixture was then analyzed by the nano-flow LC-tandem (MS/MS) system (32,33) equipped with a genome-oriented database search engine Ariadne (31). We used the genome database of Homo sapiens (reference assembly version GRCh37 obtained from ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/) under the following parameters: maximum number of missed cleavages, 0; variable modification parameter, one methylation per RNA fragment for any residue; RNA mass tolerance, ±20 ppm; and MS/MS tolerance, ±750 ppm.…”

Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly

“…Gemin5 (G5-WT-HEF)-associated snRNAs were separated by 8 M urea/9% (w/v) PAGE followed by detection with SYBR Gold staining (Invitrogen) and in-gel digestion with RNase T1 (Taoka et al 2010). SmB-associated RNAs were separated on a PLRP-S 300 Å column (2-mm I.D.…”

Friend of Prmt1, FOP is a Novel Component of the Nuclear SMN Complex Isolated Using Biotin Affinity Purification

“…In gel RNase digestion was done principality by the method described [33]. Briefly, gel pieces containing RNA bands were excised from gels, cut into small pieces, and dried under vacuum.…”

“…We also recovered several small RNA components from the nuclear SMN complexes isolated under the high salt condition ( Figure 2D), and identified the RNAs by the LC-MS/MS system equipped with a genome-oriented database searching engine Ariadne [33,35,36]. This analysis identified a number of known RNA components of the SMN complexes, U1, U3, and U4 snRNAs ( Figure 2D, supplementary Table 2), showing that our preparation of the nuclear SMN complexes in fact contained UsnRNAs.…”