Carrocci Tucker J, Zoerner Douglas M, Paulson Joshua C, Hoskins Aaron A
Department of Biochemistry, U. Wisconsin-Madison, Madison, WI 53706, USA.
Nucleic Acids Res. 2017 May 5;45(8):4837-4852. doi: 10.1093/nar/gkw1349.
RNA and protein components of the spliceosome work together to identify the 5΄ splice site, the 3΄ splice site, and the branchsite (BS) of nascent pre-mRNA. SF3b1 plays a key role in recruiting the U2 snRNP to the BS. Mutations in human SF3b1 have been linked to many diseases such as myelodysplasia (MDS) and cancer. We have used SF3b1 mutations associated with MDS to interrogate the role of the yeast ortholog, Hsh155, in BS selection and splicing. These alleles change how the spliceosome recognizes the BS and alter splicing when nonconsensus nucleotides are present at the -2, -1 and +1 positions relative to the branchpoint adenosine. This indicates that changes in BS usage observed in humans with SF3b1 mutations may result from perturbation of a conserved mechanism of BS recognition. Notably, different HSH155 alleles elicit disparate effects on splicing: some increase the fidelity of BS selection while others decrease fidelity. Our data support a model wherein conformational changes in SF3b1 promote U2 association with the BS independently of the action of the DEAD-box ATPase Prp5. We propose that SF3b1 functions to stabilize weak U2/BS duplexes to drive spliceosome assembly and splicing.
剪接体的RNA和蛋白质成分共同作用，以识别新生前体mRNA的5΄剪接位点、3΄剪接位点和分支位点（BS）。SF3b1在将U2 snRNP招募到分支位点中起关键作用。人类SF3b1中的突变与许多疾病有关，如骨髓增生异常综合征（MDS）和癌症。我们利用与MDS相关的SF3b1突变来研究酵母直系同源物Hsh155在分支位点选择和剪接中的作用。这些等位基因改变了剪接体识别分支位点的方式，并在相对于分支点腺苷的-2、-1和+1位置存在非共识核苷酸时改变剪接。这表明在具有SF3b1突变的人类中观察到的分支位点使用变化可能是由于分支位点识别保守机制的扰动所致。值得注意的是，不同的HSH155等位基因对剪接产生不同的影响：一些增加了分支位点选择的保真度，而另一些则降低了保真度。我们的数据支持一个模型，其中SF3b1的构象变化促进U2与分支位点的结合，而不依赖于DEAD-box ATPase Prp5的作用。我们提出，SF3b1的功能是稳定弱的U2/分支位点双链体，以驱动剪接体组装和剪接。
