My lab studies the process of mRNA biogenesis and export through nuclear pores, and the importance of nuclear architecture in gene expression. Another topic concerns the role of non-coding antisense RNAs in transcriptional gene silencing. Current research can be divided into 3 variations on the theme RNA:
- Role of ubiquitin in the regulation of mRNP dynamics and export through nuclear pores.
- Functional relationship between gene expression and intra-nuclear gene positioning: role of nuclear pores in transcription regulation?
- Importance of non-coding and antisense RNAs in epigenetic control of gene expression through RNAi independent mechanisms.
We use the yeast S. cerevisiae as a model system. Commonly used approaches include synthetic lethal, high-copy suppressor and two-hybrid screens, chromatin immunoprecipitation (ChIP), TAP-tag protein purification, in vitro protein interactions, transcriptome profiling, RNA analyses, RNA and gene localizations by in situ hybridization and live immunofluorescence techniques.
I. Role of ubiquitination in the regulation of mRNP dynamics and export
The different steps involved in the biogenesis and translocation of mRNAs through nuclear pores (NPC) are tightly linked. RNA polymerase II plays a central role in all these events, as it mediates the recruitment of factors involved in mRNA processing and export. All these molecular couplings are monitored by surveillance mechanisms ensuring that only fully mature and functional mRNP particles reach the cytoplasm. Our previous studies have focused on the evolutionarily conserved export receptor Mex67 and on the adaptor proteins mediating the recruitment of Mex67 to to transcribing genes and nascent mRNPs (Gwizdek, Iglesias et al., 2006; Dieppois et al., 2006). Our current work addresses the role of ubiquitin and specific E3 ligases in the co-transcriptional assembly of export competent mRNP complexes, whether different E3 ligases act in distinct export pathways and whether mRNP rearrangements may be linked to quality control mechanisms (Iglesias and Stutz, 2008; Figure 1).
Figure 1: Functional coupling and mRNP dynamics along the mRNA export pathway. Current work focuses on the ubiquitination of various export factors including the adaptor protein Yra1. Modification of Yra1 by the ubiquitin E3 ligase Tom1 results in its dissociation from the mRNP before export. This event may occur in association with Mlp1/2 at the pore and act as a license for export.
II. Functional relationship between transcription and the nuclear periphery: role of nuclear pores in transcription regulation?
A number of genes relocate to the nuclear periphery upon transcription activation. This association involves early transcription factors, as well elongation factors and mRNP components (Dieppois et al., 2006). Gene tethering to the nuclear periphery is proposed to positively influence transcription, but the molecular basis of this effect is poorly understood.
Our earlier work indicated that the nuclear pore associated Mlp1/2 proteins have been implicated in mRNA biogenesis and surveillance (Vinciguerra et al., 2005). These perinuclear proteins also anchor Ulp1, the major SUMO isopeptidase. Many nuclear proteins involved in DNA metabolism and transcription are modified by SUMO, but the exact role and regulation of this post-translational modification are still poorly defined.
Recently, we observed that loss of Mlp proteins or delocalization of Ulp1 from the nuclear periphery result in increased transcription activation kinetics. Our current working model aims at testing whether the vicinity of the pore promotes transcription by facilitating desumoylation of DNA bound transcription repressors and activators by Ulp1 (Figure 2).
Figure 2: Tup1 (and Ssn6?) transcription repressors are sumoylated, and their desumoylation by Ulp1 may promote fast relief of transcription repression and SAGA dependent activation at the NPC.
III. Role of non-coding antisense RNAs in epigenetic control of gene expression through RNAi independent mechanisms
Recent tiling array analyses show that eukaryotic genomes, including S. cerevisiae, are much more widely transcribed than expected and produce many intergenic or antisense non-coding RNAs, many of which are unstable and rapidly degraded by the nuclear exosome (Xu et al., 2009). Whether and how these non-coding RNAs participate in the regulation of gene expression is under intense investigation.
Small interfering (Si) RNAs have been implicated in transcriptional gene silencing (TGS) in S. pombe and higher eukaryotes. This process is unlikely to exist in S. cerevisiae, as this organism lacks all major components of the RNAi machinery. However, our work has revealed the existence in S. cerevisiae of alternate gene silencing mechanisms that depend on long non-coding RNAs. We have shown that loss of the exosome component Rrp6 results in PHO84 anti-sense RNA stabilization and repression of PHO84 sense transcription. Notably, anti-sense accumulation and PHO84 silencing also occur in chronologically aged cells, suggesting that Rrp6 activity is regulated in response to physiological changes. Importantly, the stabilization of PHO84 anti-sense RNAs is paralleled by the recruitment of the histone deacetylase Hda1 and deacetylation of the PHO84 promoter resulting in PHO84 gene silencing (Camblong et al., 2007, Figure 3). Our more recent data show that PHO84 anti-sense transcripts also induce TGS in trans, in a mechanism independent of Hda1. Thus, TGS in cis and trans operate through distinct pathways (Camblong et al., 2009).
To address the generality of this regulation and decipher underlying mechanisms, our project aims at identifying other genes regulated like PHO84 using genome wide approaches, as well as developing genetic screens to identify factors involved in antisense production or mediating the effects of non-coding RNAs on chromatin both in cis and trans. Our studies on budding yeast may broaden our understanding of non-coding RNAs in gene regulation in higher eukaryotes, as similar RNAi-independent mechanisms probably exist in metazoa.
Figure 3: Antisense stabilization during aging or following loss of Rrp6 is accompanied by Hda1 recruitment and histone deacetylation at the PHO84 promoter.