Research interests


Posttranscriptional control of gene expression

The activity of all genes is controlled in the nucleus at the level of transcription, the process by which a messenger RNA copy is generated from the DNA template. In addition, many genes are regulated at the posttranscriptional level, e.g. by mechanisms that control the translation efficiency and degradation rate of the mRNA in the cytoplasm. Our main focus is a group of unstable mRNAs that contain AU-rich elements. We investigate how RNA-binding proteins regulate the stability of these mRNAs, and through which pathway the mRNA is degraded. We also study the assembly of cytoplasmic processing-bodies and stress granules, sites at which mRNAs are silenced.

 

mRNA decay mediated by the AU-rich element (ARE)

Controlling the rate at which an mRNA is degraded is an important mechanism that allows cells to regulate gene expression at the posttranscriptional level. The most abundant group of mRNAs subjected to this mode of regulation are those containing an AU-rich element (ARE) in the 3' untranslated region (UTR). The presence of an ARE in a particular transcript inhibits translation of the mRNA and induces ARE-mediated mRNA decay (AMD). Potent AREs have been identified in the 3'UTRs of growth factors (e.g. GM-CSF, IL-3), cytokines (e.g. TNF-alpha, IL-2, IL-6), pro-inflammatory proteins (e.g. COX-2, MMP-13), and proto-oncogenes (e.g. c-myc, c-fos). AMD is a highly conserved regulatory mechanism that functions in yeast, trypanosomes, drosophila cells and virtually all mammalian cell lines. Thus, AMD is a general mechanism that regulates gene expression in most, if not all eukaryotic cells. Based on the frequency of AU-rich sequences in the human genome database, it has been estimated that 5-10% of all mRNAs may contain AREs. We use functional approaches such as RNA-immunoprecipitation followed by microarray analysis to explore the spectrum of mRNAs regulated by AREs.

 

The TTP/BRF family of ARE binding proteins

AREs are recognized by a variety of RNA binding proteins that influence the stability or translation efficiency of their target mRNAs. Our work focusses on the TTP/BRF family of zinc-finger proteins, which bind to certain AREs and induce rapid degadation of the mRNA. TTP/BRF proteins function as linkers between the mRNA and the general RNA decay machinery (Figure 1). The first step initiated by TTP/BRF appears to be deadenylation of the mRNA. Our recent work has shown that among the many deadenylases present in the cell, Caf1 has a major role in removing poly-A tails from ARE-containing mRNAs. TTP further interacts with the exosome that degrades RNA in the 3'-5' direction, and with the decapping/Xrn1 complex that degrades RNA in the 5'-3' direction. Cooperation with the RNA-induced silencing complex (RISC) may also contribute to controlling the translation and decay rate of ARE-mRNAs. An ongoing project in our lab is to screen for additional factors that are required for AMD.

Figure 1. The rapid degradation of mRNAs containg AREs requires RNA binding proteins such as TTP/BRF1. Upon binding to the ARE, TTP/BRF activate deadenylation of the mRNA. TTP/BRF further interact with the exosome responsible for 3'-5' decay, and with the decapping/Xrn1 complex responsible for 5'-3' decay. Interaction with the RISC complex may contribute to AMD. Proteins associated with P-bodies are depicted in red.


Regulation of ARE-mediated mRNA decay (AMD)

Activation of the immune system involves a rapid and transient production of cytokines. Many cytokine mRNAs contain AREs, and their expression is under control of the RNA-binding protein TTP. In unstimulted cells, these mRNAs are rapidly degraded. When the immune system is stimulated, transcription of cytokine mRNAs is activated in the nucleus, and the cytokine mRNAs are stabilized in the cytoplasm. The signal for stabilizing these mRNAs is mediated via the p38-MAPK - MK2 kinase pathway. Our previous work has shown that TTP is an important target of MK2 (Figure 2). MK2 phosphorylates TTP at serine 52 and 178, which leads to binding of 14-3-3 adaptor proteins. As a consequence, the activity of TTP is reduced and cytokine mRNAs are stabilized. The phosphatase PP2A acts as an antagonist of MK2 by dephosphorylating and thereby activating TTP. We are currently investigating how the phosphorylation of TTP regulates its activtiy.


Figure 2. Cytokine mRNA decay is regulated by phosphorylation of TTP. In the unphosphorylated state, TTP binds to the ARE and promotes rapid degradation of cytokine mRNAs. Phosphorylation by MK2 leads to binding of 14-3-3 adaptor proteins and inhibition of TTP activity. The phosphatase PP2A acts as an antagonist of MK2 by dephosphorylating TTP and activating mRNA decay.

 

Processing bodies: sites of translational suppression and mRNA decay

Processing (P)-bodies are small cytoplasmic foci that contain many proteins of the RNA decay machinery including deadenylases, the decapping enzymes Dcp1/Dcp2 and the 5'-3' exonuclease Xrn1. Since mRNA decay intermediates accumulate in P-bodies after inhibition of the RNA decay machinery, P-bodies are believed to be the actual sites where deadenylation, decapping and 5'-3' mRNA decay occurs. We have found that the ARE-binding proteins TTP, BRF1 and BRF2 also colocalize with P-bodies (Figure 3A), indicating that P-bodies are important for AMD. In addition, mRNAs translationally silenced by micro-RNAs are also recruited to P-bodies. This points to a dual role of P-bodies as sites of translational suppression and mRNA decay. A goal in our lab is to identify novel P-body proteins and charaterize their function.


Figure 3. (A) In transfected COS7 cells, HA-tagged TTP (red) colocalizes with P-bodies (PB, marked by Dcp1a in blue). (B) Stress granules (SG, marked by TIA1 in green) are induced after treatment with the mitochondiral inhibitor FCCP, and appear juxtaposed to P-bodies. (C) In cells transfected with HA-TTP, P-bodies are tightly associated with stress granules.

 

Stress granules: accumulation of translationally repressed mRNA

Stress granules represent a different cytoplasmic structure that only appears in cells exposed to environmental stress. Stress granules contain large amounts of untranslated mRNA that accumulates during stress-induced shut-down of protein synthesis. Although stress granules are distinct from P-bodies, they often appear juxtaposed (Figure 3B). We found that the overexpression of TTP, BRF1 and BRF2 further enhances the close physical association between stress granules and P-bodies (Figure 3C), which indicates a functional link between the two compartments. We have also shown that the recruitment of TTP to stress granules is regulated by the same phosphorylation events that inhibit TTP activity: phosphorylation at serine 52 and 178 prevents TTP from associating with stress granules. We are further exploring the connection between P-bodies and stress granules with the aim of better understanding how the translation and degradation of mRNAs is coordinately regulated.