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Related Experiment Videos

Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking.

Rebecca Hartley1, Valerie Le Meuth-Metzinger, H Beverley Osborne

  • 1Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. rebecca-hartley@uiowa.edu

BMC Molecular Biology
|June 18, 2002
PubMed
Summary

This study introduces an improved UV-crosslinking method for identifying novel RNA-binding proteins. By radiolabeling RNA with all four nucleotides, researchers can detect proteins that bind to uridine-poor sequences, enhancing gene expression studies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Post-transcriptional gene regulation involves cis-elements and trans-acting factors.
  • Genome-wide analyses aim to identify novel trans-acting factors and cis-regulatory elements for understanding coordinated gene expression.
  • Traditional UV-crosslinking uses uridine for RNA radiolabeling, potentially missing proteins binding to uridine-poor sequences.

Purpose of the Study:

  • To evaluate UV-crosslinking with RNA substrates radiolabeled with all four ribonucleotides.
  • To screen for novel sequence-specific RNA-binding proteins.

Main Methods:

  • UV-crosslinking analysis was performed using RNA substrates derived from Xenopus laevis maternal mRNAs (Eg and c-mos 3'UTRs).
  • RNA substrates were radiolabeled with each of the four ribonucleotides (CTP, GTP, ATP, UTP).

Related Experiment Videos

  • Binding sites were localized using specific RNA fragments.

    • Main Results:

    • Specific UV-crosslinking signals were obtained, with some corresponding to known proteins.
    • A novel 90 kDa protein was detected binding to the c-mos 3'UTR when RNA was radiolabeled with CTP and GTP, but not UTP.
    • The binding site for the 90 kDa protein was mapped to a 59-nucleotide region of the c-mos 3'UTR.

    Conclusions:

    • Radiolabeling with all four nucleotides in UV-crosslinking enhances the detection of RNA-binding proteins, particularly for uridine-poor binding sites.
    • This method is effective for both long and short RNAs and does not require prior knowledge of the cis-acting sequence.
    • The approach is suitable for high-throughput screening of RNA-binding proteins.