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及正常基因表达至关重要。在后生动物中，结合经典组蛋白mRNA 3'端的茎环结合蛋白（Stem-loop binding protein, SLBP）是组蛋白生物合成的关键因子。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，该过程依赖于苏氨酸60（Thr 60）和苏氨酸61（Thr 61）位点的两处磷酸化修饰。此前本团队研究表明，与谷胱甘肽S-转移酶（Glutathione S-transferase, GST）融合的SLBP片段（氨基酸残基51–108）足以模拟SLBP在S期晚期（S/G2期）的降解过程。本研究中，我们以该融合蛋白作为诱饵蛋白进行下拉实验，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。后续通过免疫共沉淀实验，我们进一步验证了全长SLBP与DCAF11、Cul4A之间的相互作用。同时我们发现，DCAF11无法与苏氨酸61突变为丙氨酸（Thr61/Ala）的突变体SLBP结合，该突变体在S期结束时不会被降解。通过异位表达实验与小干扰RNA（siRNA）实验，我们证实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，S/G2期稳定的Thr61/Ala突变体SLBP的异位表达对细胞的毒性显著更强。综上，本研究得出结论：CRL4-DCAF11复合物可介导S期结束时SLBP的降解，且该降解过程对于细胞存活至关重要。