Structural biology of pre-mRNA 3'-end processing

mRNA 前体 3 端加工的结构生物学

• 批准号: 7656634
• 负责人: LIANG TONG
• 金额: $ 30.22万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7656634
• 关键词: Address; Binding; Biochemical; Biological; Biological Assay; C-terminal; Cell Nucleus; Cleavage Stimulation Factor; Complex; Crystallography; Cytoplasm; Data; Elements; Endoribonucleases; Event; Fibrinogen; Human; Hydrolysis; Individual; Ions; Knowledge; Lactamase; Mediating; Molecular; Mus; Mutagenesis; Mutate; Poly(A) Tail; Polyadenylation; Polynucleotide Adenylyltransferase; Positioning Attribute; Process; Property; Proteins; RNA; RNA Recognition Motif; RNA Splicing; Reaction; Research Personnel; Role; Sampling; Sequence Homologs; Site; Specificity; Staging; Structure; Yeasts; Zinc; analytical ultracentrifugation; base; endonuclease; endoribonuclease; interest; mRNA Precursor; mutant; numb protein; protein protein interaction; reconstitution; research study; structural biology; yeast two hybrid system

DESCRIPTION (provided by applicant): Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo co-transcriptional processing in the nucleus before they can function as mRNAs in the cytoplasm. The processing events include 5' capping, splicing, and 3' polyadenylation. The 3' polyadenylation of pre-mRNAs occurs in two steps - endonucleolytic cleavage of the pre-mRNA at a specific site near its 3'-end and then the addition of the poly(A) tail. While the cleavage reaction may appear to be simple biochemically, it requires a large number of protein factors for its execution, including the cleavage and polyadenylation specificity factor (CPSF) complex, the cleavage stimulation factor (CstF) complex, cleavage factors I and II, and poly(A) polymerase (PAP). The CPSF complex contains four subunits, CPSF-30, CPSF-73, CPSF-100, and CPSF-160, and the CstF complex contains three subunits, CstF-50, CstF-64, and CstF-77. Despite the characterization of this large number of proteins that are required for the cleavage reaction, the identity of the endonuclease that actually catalyzes the hydrolysis is currently unknown. Recent evidence suggests CPSF-73 could be the endonuclease for the cleavage reaction, although no direct experimental evidence demonstrating this activity is available. Moreover, there is currently little structural information on these important proteins. Only the structures of PAP and the RNA binding domain of CstF-64 are known. To fill this gap in our knowledge, we have recently determined the crystal structures of human CPSF-73, yeast CPSF-100, murine CstF-77, and carried out preliminary biochemical and mutagenesis studies to assess the information from the structures. These initial results set the stage for further biochemical, biophysical, and structural studies on these proteins with crucial roles in pre-mRNA 3'-end processing.

描述（由申请人提供）：大多数真核信使RNA前体（pre-mRNAs）必须在细胞核中进行共转录加工，然后才能在细胞质中发挥mRNAs的功能。加工事件包括5'加帽、剪接和3'聚腺苷酸化。前体mRNA的3'聚腺苷酸化分两步发生-前体mRNA在其3'端附近的特定位点处的核酸内切裂解，然后添加聚腺苷酸尾。虽然切割反应在生物化学上可能看起来很简单，但它需要大量的蛋白质因子来执行，包括切割和多聚腺苷酸化特异性因子（CPSF）复合物、切割刺激因子（CstF）复合物、切割因子I和II以及多聚（A）聚合酶（PAP）。CPSF复合物含有四个亚基，CPSF-30、CPSF-73、CPSF-100和CPSF-160，CstF复合物含有三个亚基，CstF-50、CstF-64和CstF-77。 尽管对切割反应所需的大量蛋白质进行了表征，但目前尚不清楚实际催化水解的内切核酸酶的身份。最近的证据表明CPSF-73可能是切割反应的内切核酸酶，尽管没有直接的实验证据证明这种活性。此外，目前关于这些重要蛋白质的结构信息很少。只有PAP的结构和CstF-64的RNA结合结构域是已知的。为了填补我们知识中的这一空白，我们最近确定了人CPSF-73、酵母CPSF-100、鼠CstF-77的晶体结构，并进行了初步的生物化学和诱变研究以评估来自结构的信息。这些初步结果为进一步研究这些蛋白质的生物化学、生物物理学和结构奠定了基础，这些蛋白质在前mRNA 3 '端加工中起着关键作用。