Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

Abstract

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF⁶⁵) binds to the polypyrimidine (Py) tract preceding the 3′ splice site, while the 35-kDa subunit (U2AF³⁵) contacts the conserved AG dinucleotide at the 3′ end of the intron. It has been shown that the interaction between U2AF³⁵ and the 3′ splice site AG can stabilize U2AF⁶⁵ binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF³⁵ has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF⁶⁵ binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF⁶⁵. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3′ splice site AG, ESE) responsible for the U2AF³⁵ dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF³⁵dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF³⁵function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF³⁵ activity and because a truncation mutant of U2AF³⁵ consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF³⁵ can be explained in terms of enhanced U2AF⁶⁵ binding, other activities of U2AF³⁵ do not correlate with increased cross-linking of U2AF⁶⁵ to the Py tract. Collectively, the results argue that interaction of U2AF³⁵ with a consensus 3′ splice site triggers events in spliceosome assembly in addition to stabilizing U2AF⁶⁵binding, thus revealing a dual function for U2AF³⁵ in pre-mRNA splicing.