RNA polymerase II (RNAPII) transcribes all protein-encoding genes in eukaryotes and is the endpoint target for virtually all cell regulatory pathways. The overall aim of our research is to understand the basic mechanisms underlying RNAPII transcription, but in particular the mechanism and factors governing transcript elongation. For example, we strive to understand what happens when RNAPII arrests because of obstacles such as DNA damage or chromatin structure. In our work, we use a combination of biochemical and genetic approaches in yeast, Drosophila, and, increasingly, in human cells.
A detailed insight into the basic mechanisms of transcript elongation will help make it possible to understand human diseases, such as cancer, and hopefully in time to learn how to treat them.
The active, elongating form of RNAPII is tightly associated with chromatin. Remarkably, although the active forms of factors involved in other DNA-related processes such as DNA replication, repair, and recombination are also associated with chromatin, proteins are rarely purified specifically from this source. We developed a protocol for the isolation of chromatin-associated factors and used it to identify proteins interacting with the actively engaged form of human RNA polymerase II (RNAPII) (Aygün et al., 2008).
Surprisingly, beside a plethora of expected protein partners, one of the RNAPII-associated factors was a DNA helicase called RECQL5 (Figure 1). Members of the highly conserved RECQ family of DNA helicases play key roles in the maintenance of genome stability in all organisms examined. They are thought to act at the replication-recombination interface to suppress undesired recombination events that may occur due to stalled or damaged DNA replication forks. Loss-of-function mutation in 3 members of the human RecQ family of helicases, namely BLM, WRN and RECQ4, have been directly associated with genetic diseases that are characterised by premature aging and predisposition to various types of tumors due to increased genomic instability.
Figure 1. Mass spectrometric analysis of a human RNAPII complex purified from chromatin. a)outline of the purification procedure. B and C, equal amounts of the anti-FLAG chromatography eluates from control (Mock), nucleoplasm. b) and chromatin fractions. c) were fractionated by 4-12% SDS-PAGE and analyzed by silver staining. Asterisks indicate background protein bands that are also present in the control purification. Proteins identified by mass spectrometric analysis are labelled to the right of the respective protein bands.
We found that the RECQL5-RNAPII interaction is direct and mediated by the RPB1 subunit of RNAPII, and that RECQL5 is the only member of the human RECQ family of helicases that associates with RNAPII. The RNAPII-RECQ5 interaction represents an unexpected connection between transcription and genomic stability, possibly required for suppressing transcription-associated DNA recombination (Aygün et al., 2008). Our future work will be focused on further defining the RNAPII-RECQL5 connection and its functional importance in this and other processes.
The small protein-modifier ubiquitin is attached to innumerable target proteins. The final outcome of protein poly-ubiquitylation is often proteasome-mediated proteolysis, meaning that 'proof-reading' of ubiquitylation by ubiquitin proteases (UBPs) is likely to be crucial for proper regulation. Transcriptional arrest can trigger ubiquitin-mediated proteolysis of RNA polymerase II (RNAPII), so a UBP reversing RNAPII ubiquitylation might be expected.
We found that Ubp3 de-ubiquitylates RNAPII in the yeast Saccharomyces cerevisiae (Kvint et al., 2008). Indeed, genetic characterisation of cells lacking the UBP3 gene is consistent with a role for Ubp3 in transcript elongation, and Ubp3 can be purified with RNAPII, Def1, and the elongation factors Spt5 and TFIIF.
This Ubp3 complex de-ubiquitylates both mono- and poly-ubiquitylated RNAPII in vitro, and cells lacking the UBP3 gene have elevated levels of ubiquitylated RNAPII in vivo. Moreover, RNAP II is degraded faster in a ubp3 mutant after UV-irradiation. We previously proposed that problems posed by damage-arrested RNAPII are resolved either by removing the damage, or degrading the polymerase. In agreement with this, cells with compromised DNA repair are better equipped to survive UV damage when UPB3 is deleted (Kvint et al., (2008) Mol Cell 30: 498-506).
Several years ago, we isolated the Elongator complex as a component of the elongating form of RNAPII. It is now known to function in diverse cellular processes, such as RNA polymerase II transcriptional elongation and tRNA modification. The Elp3 subunit of Elongator possesses a C-terminal histone acetyltransferase (HAT) motif and an N-terminal sequence that resembles an iron-sulfur (FeS) cluster. The HAT domain is well characterised, but the role of the FeS cluster is unknown, although one report previously proposed that it might be involved in catalyzing histone demethylation (Chinenov Y. Trends Biochem Sci 2002 27: 115-117).
We investigated the importance and function of the yeast Elp3 FeS cluster by a combination of genetic and biochemical means (Greenwood et al., (2009) J Biol Chem 281: 141-149). To minimise oxidation of the Elp3 FeS cluster during purification, we also developed a novel tandem-affinity tag and an accompanying isolation procedure that enables purification of tagged proteins to virtual homogeneity within a few hours of cell disruption.
Our results failed to support a role for Elongator in histone demethylation. Moreover, we found that FeS cluster integrity is not required for the HAT or RNA-binding activities of Elongator. However, a fully functional FeS cluster is required for Elongator integrity and for the association of the complex with its accessory factors Kti11 and Kti12.
In contrast, the association of Elongator with RNAPII in chromatin is unaffected by FeS cluster mutations. Together, these data supported the idea that the Elp3 FeS cluster is essential for normal Elongator function in vivo primarily as a structural, rather than catalytic, domain (Greenwood et al., J Biol Chem. 2008; doi:10.1074/jbc. M805312200).
For a list of refereed research papers, see Publications (in navigation on left).
