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Transient transcriptome sequencing. development and applications in human cells
Transient transcriptome sequencing. development and applications in human cells
Transcription is one of the most important processes in life. Correct timing and regulation of transcription is responsible for cellular development, identity, adaptivity to environmental cues, differentiation and many other processes. Dysregulation of transcription can lead to the development of cancer and other diseases. The transcriptome of polymerase (Pol) II consists of messenger RNA (mRNA) encoding proteins and non-coding RNA (ncRNA). Due to their short half-life ncRNAs are not easy to catch and thus, many of their functions remain elusive. We set out to develop an appropriate method in human cell lines to learn more about ncRNAs, their occurrence, regulation, and kinetics. To this end we developed transient-transcriptome sequencing (TT-seq) to detect and map transient full-length RNAs in vivo. The method is based on 4-thiouridine (4sU) sequencing, where a uridine analog is supplied to cells and incorporated into nascent RNA. Fragmentation of the RNA prior to isolation of labeled RNA is the key component of the TT-seq method. This step leads to a drastic enrichment of newly-synthesized RNAs and enables detection of unstable RNAs. We employed TT-seq in the human leukemic cell line K562 and could detect and map thousands of intronic RNAs, ncRNA classes such as enhancer RNAs (eRNAs) and RNA downstream of the polyadenylation (pA) site. TT-seq enabled the analysis of synthesis and degradation rates by comparing the total RNA pool to the nascent labeled RNA fraction. In particular, we found that ncRNAs especially eRNAs are short-lived with a median half-life of 2 minutes. Analysis of RNAs downstream of the pA site enabled us to map human transcription termination sites. We found that termination sites are enriched for a (C/G)(2-6)A kmer followed by a (T/A)(3-6) kmer. This nucleotide composition can favor backtracking and/or pausing due to the change in the energy landscape of the RNA:DNA hybrid within Pol II. We propose that this facilitates termination by the exonuclease Xrn2 and is dependent on the presence of a pA site. To further understand eRNAs and their synthesis kinetics, we performed TT-seq in Jurkat T cells during the first 15 min of T cell activation. We found that thousands of mRNAs and ncRNAs transcripts were differentially expressed during the time course in the TT-seq samples. Interestingly, not a single transcript was significantly differentially expressed in the Total RNA-seq samples. This indicates that the sensitivity of TT-seq can also be used to revisit known pathways. We found that the expression levels as well as their change over time of the eRNA and the paired mRNA are highly correlated. In contrast to previous reports (Arner et al, 2015), showing that eRNA expression precedes its paired mRNA expression, we found simultaneous activation or downregulation of mRNA and eRNA pairs that are differentially expressed after 15 min. Taken together, we developed the new method TT-seq that enables mapping and analysis of newly synthesized RNAs and can determine synthesis and degradation rates. In addition, the new method is especially sensitive for transient RNAs and allows detection of very rapid expression changes. This method can be applied to any organism, which is able of 4sU (or 4-thiouracil; 4tU) uptake. Therefore, it can be broadly used to investigate many fascinating outstanding questions such as the mechanism of Pol II termination, ncRNAs degradation pathways or eRNA role in promoter activation.
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Michel, Margaux
2016
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Michel, Margaux (2016): Transient transcriptome sequencing: development and applications in human cells. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Transcription is one of the most important processes in life. Correct timing and regulation of transcription is responsible for cellular development, identity, adaptivity to environmental cues, differentiation and many other processes. Dysregulation of transcription can lead to the development of cancer and other diseases. The transcriptome of polymerase (Pol) II consists of messenger RNA (mRNA) encoding proteins and non-coding RNA (ncRNA). Due to their short half-life ncRNAs are not easy to catch and thus, many of their functions remain elusive. We set out to develop an appropriate method in human cell lines to learn more about ncRNAs, their occurrence, regulation, and kinetics. To this end we developed transient-transcriptome sequencing (TT-seq) to detect and map transient full-length RNAs in vivo. The method is based on 4-thiouridine (4sU) sequencing, where a uridine analog is supplied to cells and incorporated into nascent RNA. Fragmentation of the RNA prior to isolation of labeled RNA is the key component of the TT-seq method. This step leads to a drastic enrichment of newly-synthesized RNAs and enables detection of unstable RNAs. We employed TT-seq in the human leukemic cell line K562 and could detect and map thousands of intronic RNAs, ncRNA classes such as enhancer RNAs (eRNAs) and RNA downstream of the polyadenylation (pA) site. TT-seq enabled the analysis of synthesis and degradation rates by comparing the total RNA pool to the nascent labeled RNA fraction. In particular, we found that ncRNAs especially eRNAs are short-lived with a median half-life of 2 minutes. Analysis of RNAs downstream of the pA site enabled us to map human transcription termination sites. We found that termination sites are enriched for a (C/G)(2-6)A kmer followed by a (T/A)(3-6) kmer. This nucleotide composition can favor backtracking and/or pausing due to the change in the energy landscape of the RNA:DNA hybrid within Pol II. We propose that this facilitates termination by the exonuclease Xrn2 and is dependent on the presence of a pA site. To further understand eRNAs and their synthesis kinetics, we performed TT-seq in Jurkat T cells during the first 15 min of T cell activation. We found that thousands of mRNAs and ncRNAs transcripts were differentially expressed during the time course in the TT-seq samples. Interestingly, not a single transcript was significantly differentially expressed in the Total RNA-seq samples. This indicates that the sensitivity of TT-seq can also be used to revisit known pathways. We found that the expression levels as well as their change over time of the eRNA and the paired mRNA are highly correlated. In contrast to previous reports (Arner et al, 2015), showing that eRNA expression precedes its paired mRNA expression, we found simultaneous activation or downregulation of mRNA and eRNA pairs that are differentially expressed after 15 min. Taken together, we developed the new method TT-seq that enables mapping and analysis of newly synthesized RNAs and can determine synthesis and degradation rates. In addition, the new method is especially sensitive for transient RNAs and allows detection of very rapid expression changes. This method can be applied to any organism, which is able of 4sU (or 4-thiouracil; 4tU) uptake. Therefore, it can be broadly used to investigate many fascinating outstanding questions such as the mechanism of Pol II termination, ncRNAs degradation pathways or eRNA role in promoter activation.