PMID- 10524322
OWN - NLM
STAT- MEDLINE
DCOM- 19991119
LR  - 20180928
IS  - 0736-6205 (Print)
IS  - 0736-6205 (Linking)
VI  - 27
IP  - 4
DP  - 1999 Oct
TI  - Identification of ribonucleoprotein (RNP)-specific protein interactions using a
      yeast RNP interaction trap assay (RITA).
PG  - 790-6
AB  - We describe an adaptation of the yeast three-hybrid system that allows the
      reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins
      (RNPs). To build and try this system that we called RNP interaction trap assay
      (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct
      binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically
      tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins
      and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of
      rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was
      critically dependent on the presence of an appropriate Ro60 binding site on the
      recombinant RNA. The RITA assay was further used to detect (rRo60/rhY
      RNP)-binding proteins from a HeLa cell cDNA library, allowing specific
      identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than
      70% of positive clones. RITA assay may complement already available two- and
      three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo
      identification of interactions strictly dependent upon the simultaneous presence 
      of a protein and of its cognate RNA.
FAU - Bouffard, P
AU  - Bouffard P
AD  - Rheumatology Division, Faculty of Medicine, Universite de Sherbrooke, QC, Canada.
FAU - Briere, F
AU  - Briere F
FAU - Wellinger, R J
AU  - Wellinger RJ
FAU - Boire, G
AU  - Boire G
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Biotechniques
JT  - BioTechniques
JID - 8306785
RN  - 0 (DNA, Complementary)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Ribonucleoproteins)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Binding Sites
MH  - DNA, Complementary/analysis
MH  - Escherichia coli/genetics
MH  - Gene Library
MH  - HeLa Cells
MH  - Humans
MH  - *Molecular Probe Techniques
MH  - Plasmids/genetics
MH  - RNA/metabolism
MH  - Recombinant Proteins
MH  - *Ribonucleoproteins
MH  - Saccharomyces cerevisiae/*genetics
MH  - Transfection
MH  - Two-Hybrid System Techniques
EDAT- 1999/10/19 00:00
MHDA- 1999/10/19 00:01
CRDT- 1999/10/19 00:00
PHST- 1999/10/19 00:00 [pubmed]
PHST- 1999/10/19 00:01 [medline]
PHST- 1999/10/19 00:00 [entrez]
AID - 10.2144/99274rr02 [doi]
PST - ppublish
SO  - Biotechniques. 1999 Oct;27(4):790-6. doi: 10.2144/99274rr02.