Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1

Lili Wan, Jeong Kook Kim, Victoria W. Pollard, Gideon Dreyfuss

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The heterogeneous nuclear ribonucleoprotein (hnRNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Original languageEnglish
Pages (from-to)7681-7688
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number10
DOIs
Publication statusPublished - 2001 Mar 9

Fingerprint

Protein Interaction Domains and Motifs
RNA-Binding Proteins
RNA
Heterogeneous-Nuclear Ribonucleoproteins
Amino Acids
Heterogeneous-Nuclear Ribonucleoprotein Group C
Carrier Proteins
RNA Precursors
Leucine
Bacteriophages
Nucleic Acids
Proteins
Screening
RNA-Binding Motifs
Mutation
Oligomerization
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1. / Wan, Lili; Kim, Jeong Kook; Pollard, Victoria W.; Dreyfuss, Gideon.

In: Journal of Biological Chemistry, Vol. 276, No. 10, 09.03.2001, p. 7681-7688.

Research output: Contribution to journalArticle

@article{7d7565bba6c040e7a05d9709db0a9d66,
title = "Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1",
abstract = "The heterogeneous nuclear ribonucleoprotein (hnRNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.",
author = "Lili Wan and Kim, {Jeong Kook} and Pollard, {Victoria W.} and Gideon Dreyfuss",
year = "2001",
month = "3",
day = "9",
doi = "10.1074/jbc.M010207200",
language = "English",
volume = "276",
pages = "7681--7688",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "10",

}

TY - JOUR

T1 - Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1

AU - Wan, Lili

AU - Kim, Jeong Kook

AU - Pollard, Victoria W.

AU - Dreyfuss, Gideon

PY - 2001/3/9

Y1 - 2001/3/9

N2 - The heterogeneous nuclear ribonucleoprotein (hnRNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

AB - The heterogeneous nuclear ribonucleoprotein (hnRNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of ∼80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

UR - http://www.scopus.com/inward/record.url?scp=0035831519&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035831519&partnerID=8YFLogxK

U2 - 10.1074/jbc.M010207200

DO - 10.1074/jbc.M010207200

M3 - Article

C2 - 11113151

AN - SCOPUS:0035831519

VL - 276

SP - 7681

EP - 7688

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 10

ER -