Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry.
Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.
Ajuh
P
P
Department of Biochemistry, The University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Kuster
B
B
Panov
K
K
Zomerdijk
J C
JC
Mann
M
M
Lamond
A I
AI
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017381:Spliceosomes / Q000378:metabolism
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C085875:cdc5 protein, S pombe; D000255:Adenosine Triphosphate