Periodic Reporting for period 1 - uTSSreg (Regulation of mammalian genes by new classes of promoter proximal transcription start sites)
Periodo di rendicontazione: 2020-04-01 al 2022-03-31
The development of high-throughput sequencing methods has revealed that mammalian genomes have complex transcription patterns that generate far more spurious transcripts than RNA molecules from conventional protein-coding genes. However, the extent and content of the information encoded by such extensive transcription and the functions that the generated noncoding RNA can perform remain to be understood. RNA exosomes are evolutionarily conserved multisubunit complexes of 9 core subunits, including RRP40, with 3 '-5' exo- and endo- nucleolytic activities and in many cases, have been shown to eliminate spurious transcripts from the nucleus rapidly.
Interestingly, unpublished data recently from my lab shows that there are multiple transcription start sites (TSSs) in the promoter region, many of which produce exosome-sensitive products in hundreds of cases. Therefore, there may be previously unknown transcriptional events associated with promoters that affect the regulation of gene activity. Collecting these examples and studying the mechanisms makes it possible to discover entirely new principles of gene regulation and elucidate the molecular mechanisms underlying them. Understanding these new mechanisms is also of great importance in the related fields of developmental biology and medicine.
The overarching aim of the proposed project is to locate spurious transcription events and to investigate the impact of selected cases on local gene expression. Employing rapid depletion of nuclear RNA decay activities, I will construct a comprehensive map of transcription events, which are normally 'hidden' by RNA turnover, therefore producing unstable TSSs (uTSSs).
Interestingly, unpublished data recently from my lab shows that there are multiple transcription start sites (TSSs) in the promoter region, many of which produce exosome-sensitive products in hundreds of cases. Therefore, there may be previously unknown transcriptional events associated with promoters that affect the regulation of gene activity. Collecting these examples and studying the mechanisms makes it possible to discover entirely new principles of gene regulation and elucidate the molecular mechanisms underlying them. Understanding these new mechanisms is also of great importance in the related fields of developmental biology and medicine.
The overarching aim of the proposed project is to locate spurious transcription events and to investigate the impact of selected cases on local gene expression. Employing rapid depletion of nuclear RNA decay activities, I will construct a comprehensive map of transcription events, which are normally 'hidden' by RNA turnover, therefore producing unstable TSSs (uTSSs).
AID-tagged cells were generated in HeLa and mES cells for one part of the nuclear RNA degradation complex, RRP40 and ZCCHC8 and ZFC3H1, respectively by using CRISPR/Cas9. I checked the protein depletion by Western Blot with several IAA concentrations and time. RNA was obtained from each cell, and cDNA libraries were made for CAGE-, 3'end- and regular RNA-seq.
I found several novel transcripts containing uTSS. Among them, the LIG3 and BCAS4 gene regions were selected as uTSS in HeLa cells and the Myc gene region in ES cells I established the HeLa and mouse ES cells by manipulating TSS/uTSS region by using CRISPR/Cas9. I checked their expression level with uTSS manipulation by qPCR and found that manipulating uTSS affected the expression of unstable genes. uTSS was found to be important for the expression of this unstable RNA. To further investigate the length of this ncRNA, Northern Blot was performed and found to be a very short RNA of about 500 -1000 nt in length. The expression levels of the respective host genes were checked by qPCR with the aim of investigating whether the ncRNAs affected the expression of the host genes. The results showed down-regulation the expression levels of the uTSS-manipulated host genes I conducted chromatin immunoprecipitation (ChIP) with Histone H3K4me, H3K4Me3, H3K27Ac, H3K27me3 and RNAPII / TBP / TFIIB ChIP to understand the transcriptional state of chromatin in the cells manipulated with uTSS. To address the physiological relevance of gene expression regulation by unstable TSSs, I set up the protocol for neuronal differentiation which will take one month. I confirmed the expression of NESTIN, TUJ1 and GFAP by immunostaining and the statements of markers Tuj1, Map2 and Gfap by qPCR. For uTSS-manipulated cells, differentiation is currently underway, RNA will be collected and analysed by RNA-seq and differentiation marker will be checked by qPCR and immunostaining.
I found several novel transcripts containing uTSS. Among them, the LIG3 and BCAS4 gene regions were selected as uTSS in HeLa cells and the Myc gene region in ES cells I established the HeLa and mouse ES cells by manipulating TSS/uTSS region by using CRISPR/Cas9. I checked their expression level with uTSS manipulation by qPCR and found that manipulating uTSS affected the expression of unstable genes. uTSS was found to be important for the expression of this unstable RNA. To further investigate the length of this ncRNA, Northern Blot was performed and found to be a very short RNA of about 500 -1000 nt in length. The expression levels of the respective host genes were checked by qPCR with the aim of investigating whether the ncRNAs affected the expression of the host genes. The results showed down-regulation the expression levels of the uTSS-manipulated host genes I conducted chromatin immunoprecipitation (ChIP) with Histone H3K4me, H3K4Me3, H3K27Ac, H3K27me3 and RNAPII / TBP / TFIIB ChIP to understand the transcriptional state of chromatin in the cells manipulated with uTSS. To address the physiological relevance of gene expression regulation by unstable TSSs, I set up the protocol for neuronal differentiation which will take one month. I confirmed the expression of NESTIN, TUJ1 and GFAP by immunostaining and the statements of markers Tuj1, Map2 and Gfap by qPCR. For uTSS-manipulated cells, differentiation is currently underway, RNA will be collected and analysed by RNA-seq and differentiation marker will be checked by qPCR and immunostaining.
Recently, a new rapid protein depletion system has been developed. In the past, RNAi knockdown systems were sometimes affected secondarily by long processing times. Combining this new high-speed replication system with comprehensive transcriptome analysis, we can uncover the events that initiate transcription in mammalian cells with unprecedented precision and depth. In doing so, we will identify and classify pseudo-transcription events that may regulate the expression of protein-coding genes in ways that are not yet understood. Although the molecular concepts involved in the initiation and termination of pseudo-transcription are already known, little is known about how these events affect the transcription of neighbouring genes. My project clarifies these examples and provides new information about the mechanism. Because of the fundamental questions being asked in this project and the mES cell lines involved, it is likely to have a broad impact on the fields of molecular and developmental biology. Therefore, the results of my research are valuable to the broader scientific community in molecular biology.
Indeed, so far I have first created a pipeline to determine new RNA transcription regions. This will help to determine not only the rapidly RNA-degrading transcripts that are the focus of this study, but also previously undiscovered transcripts induced by various stimuli (e.g. immune stimuli, osmotic pressure, etc.). The transcripts transcribed from uTSS were found to affect neighbouring mRNAs, and the biological significance of this is currently being further investigated. This finding suggests that single-lived RNAs, which are usually degraded, also have a function, and further developments in molecular biology are expected.
Indeed, so far I have first created a pipeline to determine new RNA transcription regions. This will help to determine not only the rapidly RNA-degrading transcripts that are the focus of this study, but also previously undiscovered transcripts induced by various stimuli (e.g. immune stimuli, osmotic pressure, etc.). The transcripts transcribed from uTSS were found to affect neighbouring mRNAs, and the biological significance of this is currently being further investigated. This finding suggests that single-lived RNAs, which are usually degraded, also have a function, and further developments in molecular biology are expected.