CHARACTERIZATION AND MOLECULAR-CLONING OF POLYPYRIMIDINE TRACT-BINDING PROTEIN - A COMPONENT OF A COMPLEX NECESSARY FOR PRE-MESSENGER-RNA SPLICING
CHARACTERIZATION AND MOLECULAR-CLONING OF POLYPYRIMIDINE TRACT-BINDING PROTEIN - A COMPONENT OF A COMPLEX NECESSARY FOR PRE-MESSENGER-RNA SPLICING
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DOI：
10.1101/gad.5.7.1237
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发表时间：
1991-07-01
影响因子：
10.5
通讯作者：
NADALGINARD, B
中科院分区：
文献类型：
--
作者：
PATTON, JG;MAYER, SA;NADALGINARD, B
α -原肌球蛋白外显子2和3以互斥的方式拼接。外显子3作为默认外显子包含在大多数细胞类型的mRNA中，而外显子2仅包含在平滑肌细胞的mRNA中。外显子3默认选择的主要决定因素是分支点/聚嘧啶束。外显子3上游的这个元件明显有效地优于外显子2上游的相应元件。为了鉴定与这一重要顺式元件结合的反式作用因子，我们使用UV交联鉴定了一种57-kD蛋白，其结合特性与顺式竞争剪接试验中的3'-剪接位点选择直接相关。该蛋白似乎与聚嘧啶束结合蛋白相同。在本报告中，我们使用从肽序列中获得的寡核苷酸来分离和测序编码57.2 kd蛋白的cDNA克隆。初级序列揭示了一个与其他rna结合蛋白具有显著同源性的新蛋白。mRNA的表达在所有被检查的组织和细胞中都被检测到，尽管其水平表现出组织特异性和发育调节。通过生化互补实验，我们发现该蛋白与一个100-kD蛋白一起作为一个大复合体的一部分存在，该复合体需要从耗尽的核提取物中恢复剪接。
Alpha-tropomyosin exons 2 and 3 are spliced in a mutually exclusive manner. Exon 3 is included as the default exon in the mRNA of most cell types, whereas exon 2 is only included in the mRNA of smooth muscle cells. The primary determinant for the default selection of exon 3 is the branchpoint/polypyrimidine tract. This element upstream of exon 3 clearly and effectively outcompetes the corresponding element upstream of exon 2. To identify trans-acting factors that bind to this important cis element, we used UV cross-linking to identify a 57-kD protein whose binding characteristics directly correlate with 3'-splice-site selection in cis-competition splicing assays. This protein appears to be identical to polypyrimidine tract-binding protein. In this report we have used oligonucleotides derived from peptide sequences to isolate and sequence cDNA clones encoding this 57.2-kD protein. The primary sequence reveals a novel protein with significant homology to other RNA-binding proteins. Expression of the mRNA is detected in all tissues and cells examined, although its levels exhibit tissue-specific and developmental regulation. Using a biochemical complementation assay, we have found that this protein, along with a 100-kD protein, exists as part of a large complex that is required to rescue splicing from depleted nuclear extracts.