Cryo-EM studies of Gene Loops and Transcription Control

HnRNPU通过形成大的ATP依赖的低聚物，以RNA依赖的方式与染色质结合，从而调节人类细胞中的染色质长程结构(Cell 2017 V169 P1214)。我们已经使用CRISPR/生长素降解技术对hnRNPUL1(一种与hnRNPU相关的蛋白质)产生了一个人类细胞系的条件基因敲除。我们已经证明，这种蛋白参与了短而稳定的RNA的产生，如非多腺苷化的SnRNAs和组蛋白mRNAs。HnRNPUL1似乎调节这些基因的转录终止。SnRNA基因转录中一个长期存在的谜团是，如果将正常的SnRNA启动子切换为异源的RNAPII启动子，则SnRNA的正常终止失败，转录继续进行，直到达到常规的多聚腺苷信号，并且SnRNA最终异常多腺化。这一长达十年的结果表明，启动者和终结者之间的对话从未得到充分的解释。在多腺苷化蛋白编码基因上，启动子和终止子区域相互作用的基因环的出现是众所周知的，尽管涉及的因素仍然不太清楚。我们认为，hnRNPUL1与hnRNPU有许多共同的生化特性，可能在SnRNAs和组蛋白mRNAs等短基因上建立局部基因环结构，为启动子和终止子之间提供连接。此外，我们还证明了hnRNPUL1与另一种蛋白FUS形成了复合体，FUS可能调节复合体的活性。我们可以在实验室中大量生产hnRNPUL1和FUS，现在我们希望利用冷冻EM技术来探索hnRNPUL1、DNA和RNA之间形成的高分子量ATP依赖低聚物的结构以及FUS如何调节这种复合体。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体的结构/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.