Cryo-EM studies of Gene Loops and Transcription Control

hnRNPU通过形成以RNA依赖性方式与染色质缔合的大ATP依赖性寡聚体来调节人细胞中的长程染色质结构（Cell 2017 v169 p1214）。我们已经使用CRISPR/生长素降解决定子技术产生了一种人类细胞系条件性敲除hnRNPUL 1，一种与hnRNPU相关的蛋白质。我们已经表明，这种蛋白质参与了短的稳定RNA的产生，如snRNA和组蛋白mRNA，它们不是聚腺苷酸化的。hnRNPUL 1似乎调节这些基因的转录终止。snRNA基因转录中的一个长期存在的谜团是，如果正常的snRNA启动子被转换为异源RNAPII启动子，则snRNA的正常终止失败，并且转录继续，直到达到常规的多聚腺苷酸化信号并且snRNA以异常的多聚腺苷酸化结束。这个长达十年的结果表明，启动子和终止子之间的相互作用从未得到充分的解释。在多聚腺苷酸化的蛋白质编码基因上，基因环的出现是众所周知的，由此启动子和终止子区域相互作用，尽管所涉及的因素也很难被表征。我们认为hnRNPUL 1与hnRNPU具有许多共同的生化特性，可能在短基因如snRNA和组蛋白mRNA上建立局部基因环结构，以提供启动子和终止子之间的连接。此外，我们已经表明，hnRNPUL 1形成一个复合物与另一种蛋白质FUS，可以调节复合物的活性。我们可以在实验室中制造大量的hnRNPUL 1和FUS，我们现在希望利用cryo-EM技术来探测hnRNPUL 1，DNA和RNA之间形成的高分子量ATP依赖性寡聚体的结构以及FUS如何调节这种复合物。我们还将使用下一代DNA测序技术，如NETseq，在体内探索这种复合物的结构/功能。

hnRNPU regulates long range chromatin architecture in human cells by forming large ATP dependent oligomers which associate with chromatin in an RNA dependent manner (Cell 2017 v169 p1214). We have generated a human cell line conditional knockout using CRISPR/auxin degron technology for hnRNPUL1, a protein related to hnRNPU. We have shown that this protein is involved in production of short stable RNAs such as snRNAs and histone mRNAs which are not polyadenylated. hnRNPUL1 appears to regulate transcription termination of these genes. A long standing enigma in snRNA gene transcription is that if the normal snRNA promoter is switched for a heterologous RNAPII promoter then normal termination of the snRNA fails and transcription continues until a conventional polyadenylation signal is reached and the snRNA ends up aberrantly polyadenylated. This decade long result, showing that the promoter and terminator talk to each other has never been adequately explained. On polyadenylated protein coding genes the occurrence of gene loops, whereby the promoter and terminator regions interact is well known though again the factors involved are poorly characterised. We believe that hnRNPUL1, which shares many biochemical properties with hnRNPU, may establish a local gene loop structure on short genes such as snRNAs and histone mRNAs to provide the connection between the promoter and terminator. Moreover we have shown that hnRNPUL1 forms a complex with another protein FUS which may regulate the activity of the complex. We can make large quantities of hnRNPUL1 and FUS in the laboratory and we now wish to utilise cryo-EM technology to probe the structure of the high molecular weight ATP dependent oligomers formed between hnRNPUL1, DNA and RNA and how FUS regulates this complex. We will also use next generation DNA sequencing technologies such as NETseq to explore the structure/function of this complex in vivo.