Ledoux Sarah, Guthrie Christine
From the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158.
From the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158
J Biol Chem. 2016 Jun 3;291(23):11954-65. doi: 10.1074/jbc.M115.710848. Epub 2016 Apr 12.
Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices.
Brr2是一种RNA依赖性ATP酶，在剪接体组装过程中负责解开U4/U6 snRNA双链体。Brr2 RNA结合通道的棘轮螺旋内的突变会导致一种称为色素性视网膜炎（RP）的退行性人类失明。这些突变对Brr2的RNA结合、解旋酶和ATP酶活性的生化影响尚未得到表征。因此，我们鉴定出了在体外可溶的最大Brr2构建体，该构建体截短了N端的前247个氨基酸（Δ247-Brr2），以表征RP突变对Brr2活性的影响。与野生型Δ247-Brr2相比，Δ247-Brr2 RP突变体在RNA结合减弱、解旋酶活性降低和ATP酶活性降低方面表现出严重程度的梯度变化。Prp8的球状C端Jab1/Mpn1样结构域增加了Δ247-Brr2在高pH下结合U4/U6 snRNA双链体的能力，并增加了Δ247-Brr2的RNA依赖性ATP酶活性和RNA解旋程度。然而，与野生型Δ247-Brr2相比，Prp8的这个结构域对Δ247-Brr2 RP突变体没有差异影响。当受到Prp8刺激时，野生型Δ247-Brr2能够在体外解开长的稳定双链体，甚至是能够紧密结合RNA的RP突变体也无法完全解开短的双链RNA。我们的数据表明，棘轮螺旋内的RP突变损害了Brr2通过RNA螺旋的易位。
