Cloning of Human 2H9 Heterogeneous Nuclear Ribonucleoproteins

doi:10.1074/jbc.272.3.1827

Cloning of Human 2H9 Heterogeneous Nuclear Ribonucleoproteins

RELATION WITH SPLICING AND EARLY HEAT SHOCK-INDUCED SPLICING ARREST*

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France and the
^§ U 242 de l'Inserm, Hôpital des Enfants, Groupe Hospitalier de la Timone, 13385 Marseille, Cedex 5, France

^¶To whom all correspondence should be addressed. Present address:
Institut National de la Santé et de la Recherche Médicale, Unité 338 de Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg, France.
Tel.: 33 3 88 45 66 09; Fax: 33 3 88 60 08 06; E-mail: fuchs{at}neurochem.u-strasbg.fr

Abstract

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45°C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H′ family. Protein 2H9 displays two consensus sequence-type RNA binding domains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H′. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

Footnotes

↵^‡ Sponsored by the Ministère de la Recherche et de l'Espace and by the Ligue contre le Cancer.
↵* This work was supported by funds of the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Centre Hospitalier Universitaire Régional, and by special grants of the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) L32610[GenBank].
↵¹ The abbreviations used are:

hnRNA

heterogeneous nuclear RNA

hnRNP

heterogeneous nuclear ribonucleoprotein

snRNP

small nuclear ribonucleoprotein

CS-RBD

consensus sequence RNA binding domain

DTT

dithiothreitol

PBS

phosphate-buffered saline

SSC

sodium chloride/sodium citrate

PAGE

polyacrylamide gel electrophoresis

mAb

monoclonal antibody

pAb

polyclonal antibody.
- Received July 23, 1996.
- Revision received September 25, 1996.