Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Summary

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Abstract

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5' end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3' end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

Introduction

In eukaryotes, RNA polymerase II-generated mRNA precursors (pre-mRNAs) undergo several maturation events in the nucleus before becoming fully functional mRNA templates for protein synthesis in the cytoplasm. One of these events is 3' end processing. For the vast majority of pre-mRNAs, 3' end processing involves cleavage coupled to polyadenylation. This two-step reaction is catalyzed by a relatively abundant complex consisting of more than 15 proteins¹. Animal replication-dependent histone pre-mRNAs are processed at the 3' end by a different mechanism in which the key role is played by U7 snRNP, a low abundance complex consisting of ....

Protocol

1. Substrate Preparation

NOTE: RNA substrates shorter than ~65 nucleotides can be synthesized chemically with biotin (B) and the photo-cleavable (pc) linker (together referred to as photo-cleavable biotin or pcB) covalently attached to the RNA 5’ end (cis configuration). RNA substrates containing significantly longer binding sites need to be generated in vitro by T7 (or SP6) transcription and subsequently annealed to a short complementary adaptor oligonucleotide containing pcB moiety at the 5’ end (trans configuration) (Figure 1).

1. For binding sites consisting o....

Results

The UV-elution method was tested with two chemically synthesized RNA substrates covalently attached at the 5' end to the pcB moiety (cis configuration): pcB-SL (Figure 1) and pcB-dH3/5m RNAs (Figure 2). The 31-nucleotide pcB-SL RNA contains a stem-loop structure followed by a 5-nucleotide single stranded tail and its sequence is identical to the 3' end of mature histone mRNA (i.e., after the cleavage of histone .......

Discussion

The method described here is straightforward and besides incorporating a photo-cleavable linker and the UV-elution step does not differ from the commonly used methods that take advantage of the extremely strong interaction between biotin and streptavidin. The UV-elution step is very efficient, typically releasing more than 75% of the immobilized RNA and associated proteins from streptavidin beads, leaving behind a high background of proteins that non-specifically bind to the beads. By eliminating this background, the UV-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Streptavidin-Agarose	Sigma	S1638-5ML
High-intensity, long-wave UV lamp	Cole-Parmer	UX-97600-00
Replacement bulb	Cole-Parmer	UX-97600-19	100 Watts Mercury H44GS-100M bulb emitting 366 nm UV light (Sylvania)
RNAs and oligonucleotides containing biotin and photo-cleavable linker in cis	Dharmacon (Lafayette, CO) or Integrated DNA Technologies, Inc. (Coralville, IA)		Requst a quote in Dharmacon
Beckman GH 3.8 swing bucket rotor	Beckman
RiboMAX Large Scale RNA Production Systems (T7 polymerase)	Promega	P1300
RiboMAX Large Scale RNA Production Systems (SP6 polymerase)	Promega	P1280
Pierce Silver Stain Kit	ThermoFisher Scientific	24612