Final Report Summary - EXTRATF (Global positioning of TFIIIC and its involvement in extra-transcriptional processes (ExtraTF))
Eukaryotic cells have developed complex protein systems to employ the information that is safely stored in its genome. One of these processes is called transcription, whereby a certain region of interest of the DNA is transcribed into RNA. Three different RNA polymerase systems are involved in the regulation of the transcription process, and the promoter determines which polymerase is required. RNA Polymerase I (Pol I) transcribes one large ribosomal RNA gene, RNA Polymerase II (Pol II) transcribes all mRNA genes and most small nuclear RNA (snRNA) genes, and RNA Polymerase III (Pol III) transcribes small RNA molecules, such as the small 5S rRNA, the U6 spliceosomal RNA and transfer RNAs (tRNAs).
tRNAs are the building blocks of proteins and it is vital that their expression is tightly regulated to avoid under or over production of protein, that might lead to diseases such as cancer. To regulate the transcription of tRNAs, Pol III requires the TFIIIC, that binds to the regulatory region within the tRNA genes and recruits the transcription factor IIIB and Pol III, that initiates transcription.
The Intra-European Fellowship “Extra TF” proposed the analysis of the transcription factor IIIC (TFIIIC) via in vivo microscopy in budding yeast. The main objectives of this proposal were to understand the intricate regulation mechanisms developed by eukaryotic cells, to correctly grow and divide. Transcription is one of the most highly regulated mechanisms in the cell, and it is regulated at different levels. Understanding this requires a multidisciplinary approach, and this proposal aims to contribute to this understanding.
With this project we studied TFIIIC with fluorescence microscopy, to understand its dynamics in the nucleus and the cytoplasm, and with functional assays, to understand how it regulates the transcription of the different RNAs. We showed that the subunits of TFIIIC exist in similar number in the nucleus and cytoplasm of a yeast cell. The affinity between the subunits is very high in the nucleus, but it is below the detection limit in the cytoplasm. This indicates that this complex mainly exists in the nucleus, where it is active. We further tested dynamics of the complex in the nucleus, by selectively degrading one subunit at a time (using so-called degron strains), and we observed that there is a special link between two of the subunits, tau138 and tau55: tau55 is relocated to the cytoplasm upon degradation of tau138. This seems to indicate that tau55 requires tau138 to stay stable in the nucleus. Additional strains were also made to study the interaction between subunits of TFIIIC, TFIIIB and Pol III. This work was done in collaboration with Prof. Michael Knop (from the ZMBH/DKFZ) and Dr. Malte Wachsmuth (Cell Biology and Biophysics, EMBL Heidelberg).
To get a more functional insight into TFIIIC, we used the six degron strains that we generated previously and tested them in a functional assay. For this, we designed two complementary assays to determine the effect on the RNA transcription by Pol III, upon degradation of subunits from TFIIIC. We established qPCR and RNA sequencing protocols to quantify the RNA transcribed by Pol III under different conditions and we assessed the effect of degrading each subunit of TFIIIC.
The qPCR results showed that the degradation of the TFIIIC subunits resulted in an increase in the transcription of tRNAs, whereas the transcription of other Pol III transcribed RNAs show no change or a decrease in expression. Our results indicate that tRNAs might undergo a different transcriptional regulation in comparison to the remaining Pol III RNA genes that require TFIIIC. This is a new and interesting finding. To confirm this and to analyze other characteristics of tRNA, such as sequence, length and splicing forms, we performed tRNA sequencing experiments. The protocol for tRNA sequencing was applied to tRNAs and optimized, as was the data analysis. The final experiments are being finished at the moment. In addition, the protocol we developed is very efficient and reproducible and we are currently preparing it for publication. The data analysis was done in collaboration with the Steinmetz group (EMBL, Heidelberg). The impact of this work can be quite significant: it has been suggested that the expression levels of tRNA can be used as biomarkers for certain diseases such as cancer and neurodegenerative diseases (reviewed in Tuller, Frontiers in Genetics). For this reason, both the library preparation protocol and the data analysis pipeline is potentially very useful to other scientists
tRNAs are the building blocks of proteins and it is vital that their expression is tightly regulated to avoid under or over production of protein, that might lead to diseases such as cancer. To regulate the transcription of tRNAs, Pol III requires the TFIIIC, that binds to the regulatory region within the tRNA genes and recruits the transcription factor IIIB and Pol III, that initiates transcription.
The Intra-European Fellowship “Extra TF” proposed the analysis of the transcription factor IIIC (TFIIIC) via in vivo microscopy in budding yeast. The main objectives of this proposal were to understand the intricate regulation mechanisms developed by eukaryotic cells, to correctly grow and divide. Transcription is one of the most highly regulated mechanisms in the cell, and it is regulated at different levels. Understanding this requires a multidisciplinary approach, and this proposal aims to contribute to this understanding.
With this project we studied TFIIIC with fluorescence microscopy, to understand its dynamics in the nucleus and the cytoplasm, and with functional assays, to understand how it regulates the transcription of the different RNAs. We showed that the subunits of TFIIIC exist in similar number in the nucleus and cytoplasm of a yeast cell. The affinity between the subunits is very high in the nucleus, but it is below the detection limit in the cytoplasm. This indicates that this complex mainly exists in the nucleus, where it is active. We further tested dynamics of the complex in the nucleus, by selectively degrading one subunit at a time (using so-called degron strains), and we observed that there is a special link between two of the subunits, tau138 and tau55: tau55 is relocated to the cytoplasm upon degradation of tau138. This seems to indicate that tau55 requires tau138 to stay stable in the nucleus. Additional strains were also made to study the interaction between subunits of TFIIIC, TFIIIB and Pol III. This work was done in collaboration with Prof. Michael Knop (from the ZMBH/DKFZ) and Dr. Malte Wachsmuth (Cell Biology and Biophysics, EMBL Heidelberg).
To get a more functional insight into TFIIIC, we used the six degron strains that we generated previously and tested them in a functional assay. For this, we designed two complementary assays to determine the effect on the RNA transcription by Pol III, upon degradation of subunits from TFIIIC. We established qPCR and RNA sequencing protocols to quantify the RNA transcribed by Pol III under different conditions and we assessed the effect of degrading each subunit of TFIIIC.
The qPCR results showed that the degradation of the TFIIIC subunits resulted in an increase in the transcription of tRNAs, whereas the transcription of other Pol III transcribed RNAs show no change or a decrease in expression. Our results indicate that tRNAs might undergo a different transcriptional regulation in comparison to the remaining Pol III RNA genes that require TFIIIC. This is a new and interesting finding. To confirm this and to analyze other characteristics of tRNA, such as sequence, length and splicing forms, we performed tRNA sequencing experiments. The protocol for tRNA sequencing was applied to tRNAs and optimized, as was the data analysis. The final experiments are being finished at the moment. In addition, the protocol we developed is very efficient and reproducible and we are currently preparing it for publication. The data analysis was done in collaboration with the Steinmetz group (EMBL, Heidelberg). The impact of this work can be quite significant: it has been suggested that the expression levels of tRNA can be used as biomarkers for certain diseases such as cancer and neurodegenerative diseases (reviewed in Tuller, Frontiers in Genetics). For this reason, both the library preparation protocol and the data analysis pipeline is potentially very useful to other scientists