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J. Biol. Chem., Vol. 277, Issue 26, 23764-23772, June 28, 2002
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From the Human splicing factors Hprp3p and Hprp4p are
associated with the U4/U6 small nuclear ribonucleoprotein particle,
which is essential for the assembly of an active spliceosome.
Currently, little is known about the specific roles of these factors in
splicing. In this study, we characterized the molecular interaction
between Hprp3p and Hprp4p. Constructs were created for expression of
Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the
Hprp3p-Hprp4p complex formed in Escherichia
coli lysates. By co-immunoprecipitation and isothermal titration
calorimetry, we demonstrated that purified Hprp3p and its mutants
containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N- or
C-terminal region did not interact with Hprp4p. In addition, by
co-immunoprecipitation, we showed that intact Hprp3p and its mutants
containing the central region interacted with Hprp4p in HeLa cell
nuclear extracts. Primer extension analysis illustrated that the
central region of Hprp3p is required to maintain the association of
Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this
Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps
other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.
Central Region of the Human Splicing Factor Hprp3p
Interacts with Hprp4p*,
§,,
,§**
Program in Lung Biology Research,
Hospital for Sick Children, and the Departments of
** Paediatrics, § Laboratory Medicine and
Pathobiology, and ¶ Medical Genetics and Microbiology, University
of Toronto, Toronto, Ontario M5G 1X8, Canada
*
This work was supported in part by an operating grant from
the Canadian Institute of Health Research (to J. H.) and by grants from the Canada Foundation for Innovation and the Ontario Innovation Fund (to J. L.).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 on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 1 and 2.
Present address: Dept. of Biochemistry and Biotechnology,
Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan.
Recipient of a scholarship award from the Canadian Cystic
Fibrosis Foundation. To whom correspondence should be addressed: Program in Lung Biology Research, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6412; Fax: 416-813-5771; E-mail: jhu@sickkids.on.ca.
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