Preparation of cooperative RNA recognition complexes for crystallographic structural studies

Chen Qiu, Aaron Goldstrohm, Traci M. Tanaka Hall

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

It is essential that mRNA-binding proteins recognize specific motifs in target mRNAs to control their processing, localization, and expression. Although mRNAs are typically targets of many different regulatory factors, our understanding of how they work together is limited. In some cases, RNA-binding proteins work cooperatively to regulate an mRNA target. A classic example is Drosophila melanogaster Pumilio (Pum) and Nanos (Nos). Pum is a sequence-specific RNA-binding protein. Nos also binds RNA, but interaction with some targets requires Pum to bind first. We recently determined crystal structures of complexes of Pum and Nos with two different target RNA sequences. A crystal structure in complex with the hunchback mRNA element showed how Pum and Nos together can recognize an extended RNA sequence with Nos binding to an A/U-rich sequence 5′ of the Pum sequence element. Nos also enables recognition of elements that contain an A/U-rich 5′ sequence, but imperfectly match the Pum sequence element. We determined a crystal structure of Pum and Nos in complex with the Cyclin B mRNA element, which demonstrated how Nos clamps the Pum-RNA complex and enables recognition of the imperfect element. Here, we describe methods for expression and purification of stable Pum-Nos-RNA complexes for crystallization, details of the crystallization and structure determination, and guidance on how to analyze protein-RNA structures and evaluate structure-driven hypotheses. We aim to provide tips and guidance that can be applied to other protein-RNA complexes. With hundreds of mRNA-binding proteins identified, combinatorial control is likely to be common, and much work remains to understand them structurally.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsAmanda E. Hargrove
PublisherAcademic Press Inc.
Pages1-22
Number of pages22
ISBN (Print)9780128168318
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Enzymology
Volume623
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

RNA
Messenger RNA
RNA-Binding Proteins
Crystal structure
Crystallization
Carrier Proteins
Cyclin B
Clamping devices
Drosophila melanogaster
Purification
Proteins
Processing

Keywords

  • Cooperative RNA binding
  • Nanos
  • PUF protein
  • Pumilio
  • X-ray crystallography

PubMed: MeSH publication types

  • Journal Article

Cite this

Qiu, C., Goldstrohm, A., & Tanaka Hall, T. M. (2019). Preparation of cooperative RNA recognition complexes for crystallographic structural studies. In A. E. Hargrove (Ed.), Methods in Enzymology (pp. 1-22). (Methods in Enzymology; Vol. 623). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.04.001

Preparation of cooperative RNA recognition complexes for crystallographic structural studies. / Qiu, Chen; Goldstrohm, Aaron; Tanaka Hall, Traci M.

Methods in Enzymology. ed. / Amanda E. Hargrove. Academic Press Inc., 2019. p. 1-22 (Methods in Enzymology; Vol. 623).

Research output: Chapter in Book/Report/Conference proceedingChapter

Qiu, C, Goldstrohm, A & Tanaka Hall, TM 2019, Preparation of cooperative RNA recognition complexes for crystallographic structural studies. in AE Hargrove (ed.), Methods in Enzymology. Methods in Enzymology, vol. 623, Academic Press Inc., pp. 1-22. https://doi.org/10.1016/bs.mie.2019.04.001
Qiu C, Goldstrohm A, Tanaka Hall TM. Preparation of cooperative RNA recognition complexes for crystallographic structural studies. In Hargrove AE, editor, Methods in Enzymology. Academic Press Inc. 2019. p. 1-22. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2019.04.001
Qiu, Chen ; Goldstrohm, Aaron ; Tanaka Hall, Traci M. / Preparation of cooperative RNA recognition complexes for crystallographic structural studies. Methods in Enzymology. editor / Amanda E. Hargrove. Academic Press Inc., 2019. pp. 1-22 (Methods in Enzymology).
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