DDB1 and CUL4 associated factor 11 (DCAF11) mediates degradation of Stem-loop binding protein at the end of S phase

In eukaryotes, bulk histone expression occurs in the S phase of the cell cycle. This highly conserved system is crucial for genomic stability and proper gene expression. In metazoans, Stem-loop binding protein (SLBP), which binds to 3′ ends of canonical histone mRNAs, is a key factor in histone biosynthesis. SLBP is mainly expressed in S phase and this is a major mechanism to limit bulk histone production to the S phase. At the end of S phase, SLBP is rapidly degraded by proteasome, depending on two phosphorylations on Thr 60 and Thr 61. Previously, we showed that SLBP fragment (aa 51–108) fused to GST, is sufficient to mimic the late S phase (S/G2) degradation of SLBP. Here, using this fusion protein as bait, we performed pull-down experiments and found that DCAF11, which is a substrate receptor of CRL4 complexes, binds to the phosphorylated SLBP fragment. We further confirmed the interaction of full-length SLBP with DCAF11 and Cul4A by co-immunoprecipitation experiments. We also showed that DCAF11 cannot bind to the Thr61/Ala mutant SLBP, which is not degraded at the end of S phase. Using ectopic expression and siRNA experiments, we demonstrated that SLBP expression is inversely correlated with DCAF11 levels, consistent with the model that DCAF11 mediates SLBP degradation. Finally, we found that ectopic expression of the S/G2 stable mutant SLBP (Thr61/Ala) is significantly more toxic to the cells, in comparison to wild type SLBP. Overall, we concluded that CRL4-DCAF11 mediates the degradation of SLBP at the end of S phase and this degradation is essential for the viability of cells.

在真核生物中，大量组蛋白的表达发生在细胞周期的S期。这一高度保守的系统对于基因组稳定性与正常基因表达至关重要。在后生动物中，茎环结合蛋白（Stem-loop binding protein, SLBP）可结合经典组蛋白mRNA的3'末端，是组蛋白生物合成过程中的关键因子。SLBP主要在S期表达，这也是将大量组蛋白合成限制在S期的核心机制。在S期结束时，SLBP会被蛋白酶体快速降解，这一过程依赖于苏氨酸60（Thr60）与苏氨酸61（Thr61）位点的两处磷酸化修饰。此前本团队的研究表明，与谷胱甘肽S-转移酶（Glutathione S-transferase, GST）融合的SLBP氨基酸残基51-108片段，足以模拟SLBP在S期晚期（S/G2期）的降解过程。本研究中，我们以该融合蛋白为诱饵开展亲和下拉实验，发现作为CRL4复合物底物受体的DCAF11可与磷酸化的SLBP片段结合。我们进一步通过免疫共沉淀实验，验证了全长SLBP与DCAF11、Cul4A（Cullin 4A）之间的相互作用。我们还发现，DCAF11无法与苏氨酸61丙氨酸（Thr61/Ala）突变的SLBP结合，这类突变体在S期结束时不会被降解。我们通过异位表达与小干扰RNA（small interfering RNA, siRNA）实验证实，SLBP的表达水平与DCAF11的表达量呈负相关，这与DCAF11介导SLBP降解的模型相符。最后我们发现，与野生型SLBP相比，异位表达S/G2期稳定的SLBP苏氨酸61丙氨酸突变体对细胞的毒性显著更强。综上，我们得出结论：CRL4-DCAF11复合物介导S期结束时SLBP的降解，且该降解过程对细胞存活至关重要。