Cryo-EM studies of Gene Loops and Transcription Control

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.