Distinct sequence motifs within the 68-kDa subunit of cleavage factor Im mediate RNA binding, protein-protein interactions, and subcellular localization.
Cleavage factor I(m) (CF I(m)) is required for the first step in pre-mRNA 3'-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68-kDa subunits. The binding of CF I(m) to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF I(m) involved in RNA binding, protein-protein interactions, and subcellular localization. CF I(m)68 has a modular domain organization consisting of an N-terminal RNA recognition motif and a C-terminal alternating charge domain. However, the RNA recognition motif of CF I(m)68 on its own is not sufficient to bind RNA but is necessary for association with the 25-kDa subunit. RNA binding appears to require a CF I(m)68/25 heterodimer. Whereas multiple protein interactions with other 3'-end-processing factors are detected with CF I(m)25, CF I(m)68 interacts with SRp20, 9G8, and hTra2beta, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF I(m)68 to the nucleus. However, CF I(m)68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found.
Dettwiler
Sabine
S
Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Aringhieri
Chiara
C
Cardinale
Stefano
S
Keller
Walter
W
Barabino
Silvia M L
SM
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D017421:Sequence Analysis; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics* / Q000378:metabolism
D021122:Protein Subunits; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C450562:cleavage factor Im, human; D039221:mRNA Cleavage and Polyadenylation Factors