It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5′ splice site (5′SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM–RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.

IκB proteins are known as the regulators of NF‐κB activity. They bind tightly to NF‐κB dimers, until stimulus‐responsive N‐terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IκBα is an unstable protein whose rapid degradation is slowed upon binding to NF‐κB, but it is not known what dynamic mechanisms control the steady‐state level of total IκBα. Here, we show clearly that two degradation pathways control the level of IκBα. Free IκBα degradation is not controlled by IKK or ubiquitination but intrinsically, by the C‐terminal sequence known as the PEST domain. NF‐κB binding to IκBα masks the PEST domain from proteasomal recognition, precluding ubiquitin‐independent degradation; bound IκBα then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IκBα protein; alteration of free IκBα degradation dampens NF‐κB activation. In addition, we find that both free and bound IκBα are similar substrates for IKK, and the preferential phosphorylation of NF‐κB‐bound IκBα is due to stabilization of IκBα by NF‐κB.

We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50) ₂ ], which exhibits little sequence resemblance to the consensus DNA target for (p50) ₂ , but binds (p50) ₂ with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50) ₂ .