Cryo-EM studies of Gene Loops and Transcription Control

hnRNPU通过形成以RNA依赖方式与染色质结合的ATP依赖性大寡聚物来调节人类细胞中的长距离染色质结构（Cell 2017 v169 p1214）。我们利用CRISPR/生长素脱粒技术对hnRNPU相关蛋白hnRNPUL1进行了人类细胞系条件敲除。我们已经证明，这种蛋白质参与了短稳定rna的产生，如snrna和组蛋白mrna，这些rna不会被聚腺苷化。hnRNPUL1似乎调控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，如果正常snRNA启动子被切换为异源RNAPII启动子，则snRNA的正常终止失败，转录继续进行，直到到达常规的多腺苷化信号，snRNA最终异常多腺苷化。这个长达十年的研究结果表明，启动子和终止子之间的相互作用从未得到充分的解释。在聚腺苷化的蛋白质编码基因上，启动子和终止子区域相互作用的基因环的发生是众所周知的，尽管所涉及的因素也没有得到很好的表征。我们认为hnRNPUL1与hnRNPU具有许多相同的生化特性，可能在snrna和组蛋白mrna等短基因上建立局部基因环结构，提供启动子和终止子之间的连接。此外，我们已经证明hnRNPUL1与另一种蛋白FUS形成复合体，可能调节复合体的活性。我们可以在实验室中制造大量的hnRNPUL1和FUS，现在我们希望利用冷冻电镜技术来探测hnRNPUL1、DNA和RNA之间形成的高分子量ATP依赖性低聚物的结构，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，在体内探索该复合体的结构/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.