Radical probing of spliceosome assembly

Charnpal S Grewal; Oliver A Kent; Andrew M MacMillan

doi:10.1016/j.ymeth.2017.06.030

Radical probing of spliceosome assembly

Methods. 2017 Aug 1:125:16-24. doi: 10.1016/j.ymeth.2017.06.030. Epub 2017 Jun 29.

Authors

Charnpal S Grewal¹, Oliver A Kent², Andrew M MacMillan³

Affiliations

¹ Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.
² Princess Margaret Cancer Centre, 101 College St., University of Toronto, Toronto, ON M5G 1L7, Canada.
³ Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada. Electronic address: [email protected].

PMID: 28669867
DOI: 10.1016/j.ymeth.2017.06.030

Abstract

Here we describe the synthesis and use of a directed hydroxyl radical probe, tethered to a pre-mRNA substrate, to map the structure of this substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly. This methodology may be adapted to the synthesis of a wide variety of modified RNAs for use as probes of RNA structure and RNA-protein interaction.

Keywords: Footprinting; Hydroxyl radical; Spliceosome assembly; pre-mRNA splicing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Crystallography, X-Ray / methods
DNA-Directed RNA Polymerases / metabolism
Hydroxyl Radical / chemistry*
Hydroxyl Radical / metabolism
Molecular Probe Techniques*
Molecular Probes / chemical synthesis*
Oligonucleotides / metabolism
RNA Precursors / chemistry*
RNA Precursors / metabolism
RNA Splicing
Spliceosomes / chemistry
Spliceosomes / metabolism*
Viral Proteins / metabolism

Substances

Molecular Probes
Oligonucleotides
RNA Precursors
Viral Proteins
Hydroxyl Radical
bacteriophage T7 RNA polymerase
DNA-Directed RNA Polymerases

Grants and funding

CIHR/Canada