Christian Kroun Damgaard

Research area: The molecular mechanisms underlying regulated mRNA decay and translation during cellular stress

Damgaard Lab

 

The broad aim of our research group is to decipher the molecular mechanisms underlying regulated mRNA decay and translation during cellular stress, processes that are often deregulated in disease.


Cellular stress re-programs gene-expression both at the transcriptional and post-transcriptional levels. Transcriptional events have traditionally received most attention in the past, but it has become increasingly clear that post-transcriptional regulation of gene-expression, such as modulation of mRNA decay rates and translation, plays an equally important role for a proper regulation of the eukaryotic transcriptome. We use human cell-lines as model systems to directly study how mRNA decay and translation is regulated by interaction with a variety of RNA-binding proteins and non-coding RNAs. We utilize cellular imaging, including single molecule mRNA fluorescent in situ hybridization (RNA-FISH), immunofluorescence and molecular biological and biochemical tools to address three major focus areas of our research:

 

1.  Translational regulation during stress

We have recently revealed that two RNA-binding proteins, coined TIA-1 and TIAR, work as important repressors of a certain class of mRNAs containing a 5'Terminal OligoPyrimidine tract (5'TOP) during amino acid starvation. Our current research efforts focus on further defining the mechanistic details by which these proteins act to repress translation during stress. These studies include purification of 5'TOP-mRNA-specific complexes, whose composition is scrutinized by mass spectrometry (MS). Other approaches include global assays, which aim at identifying novel translation events that are regulated by starvation and/or TIA-proteins and how cellular signaling regulates this process.

2.  Regulation of mRNA decay rates by RNA-binding proteins and non-coding RNAs (ncRNAs)

Many naturally labile mRNAs are regulated by elements in their 3'UTR including AU-rich elements and microRNA (miRNA) binding sites. These elements interact with a variety of AU-rich-binding proteins and/or miRNA-loaded complexes, respectively, to modulate the recruitment of mRNA decay factors in order to control the rate of mRNA decay. These regulatory networks enable the cell to rapidly adapt to stress situations and mount a proper response and ensure survival. To study the regulation of mRNA degradation rates during activation or deactivation of certain signaling pathways, we employ pulse-chase mRNA decay assays, which directly measure mRNA decay rates, and thereby remove any contribution from altered transcription. Some of our efforts specifically focus on disease-causing mRNAs such as those encoding onco-proteins and autoimmune regulators, where deregulated expression is directly associated with disease. Others include unbiased global assays, which aim at elucidating mRNA targets that are regulated during different cellular stresses. Current investigations also aim to identify long non-coding RNAs (lncRNAs) that are potentially involved the regulation of mRNA decay and translation, and to further elucidate the mechanistic modes of their action.

 

3. Context-dependent miRNA regulation

Since the discovery of regulatory non-coding RNAs, such as miRNAs, much research has been focused on post-transcriptional gene-regulation and in turn on the mechanistic details defining miRNA function. miRNA target-sites, which are usually found in 3'UTRs of target mRNAs, are notoriously hard to predict, due to the relatively short miRNA/mRNA interaction sequence. Although numerous prediction algorithms are available, most of these still fail to credibly predict in vivo target sites, since their recognition is affected by other elements and trans-acting factors. In order to better understand miRNA/mRNA interactions and to allow a more credible prediction of target sites, we are currently scrutinizing available datasets for non-miRNA-target elements that associate positively or negatively with miRNA-mediated mRNA destabilization. We furthermore wish to identify possible RNA-binding factors, which interact with these non-target elements, by biochemical purification/identification.

Figure legend:

RNA-FISH experiment showing a 5'TOP-mRNA reporter accumulating in stress granules in HeLa cells.

Click figure for enlargement.