TY - JOUR
T1 - Nuclear actin is associated with a specific subset of hnRNP A/B-type proteins
AU - Percipalle, Piergiorgio
AU - Jonsson, Andreas
AU - Nashchekin, Dmitri
AU - Karlsson, Christina
AU - Bergman, Tomas
AU - Guialis, Apostolia
AU - Daneholt, Bertil
N1 - Funding Information:
We are grateful to Lise-Marie Fjelkestam and Carina Palmberg for technical assistance. The plasmid vectors for bacterial and mammalian expression of DBP40 were kindly donated by Dr Colin R. Parrish and purified actin by Dr Uno Lindberg. We thank Dr Adrian Krainer for supplying the pET9d-hnRNP A2 and Dr Gideon Dreyfuss for providing antibodies against hnRNP A1 and hnRNP C1/C2. The work was supported by the Swedish Research Council (B 5101-879/2001 to B.D.; K 5104-20005891 to T.B.), the Human Frontier Science Program Organization, the Knut and Alice Wallenberg Foundation, and the Ingabritt and Arne Lundberg Foundation. P.P. received Research Fellowships from the European Community and the Blanceflor-Ludovisi Foundation, D.N. a Fellowship from INTAS, and A.G. a Short Term Fellowship from EMBO and a stipend from the Swedish Institute.
PY - 2002/4/15
Y1 - 2002/4/15
N2 - Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA-protein and protein-protein cross-linking experiments showed that DBP40 interacts with poly(A)+ RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.
AB - Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA-protein and protein-protein cross-linking experiments showed that DBP40 interacts with poly(A)+ RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.
UR - http://www.scopus.com/inward/record.url?scp=0037089149&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037089149&partnerID=8YFLogxK
U2 - 10.1093/nar/30.8.1725
DO - 10.1093/nar/30.8.1725
M3 - Article
C2 - 11937625
AN - SCOPUS:0037089149
VL - 30
SP - 1725
EP - 1734
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 8
ER -