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Nuclear Human RNA Decay

The RNA exosome and its network of co-factors. The exosome is comprised of 9 catalytically inert subunits (EXO9) associating with distinct ribonucleases (DIS3, DIS3L, RRP6) and co-factors depending on the sub-cellular localisation of the complex. Our laboratory has characterized essential adaptor complexes of the nuclear exosome: The Nuclear EXosome Targeting (NEXT) complex (MTR4, ZCCHC8, RBM7), the PolyA eXosome Targeting (PAXT) connection (MTR4, ZFC3H1, PABPN1) and the human TRf/Air/Mtr4 polyadenylation (TRAMP) complex (MTR4, ZCCHC7, TRF4-2). For further reading see: Lubas et al. Mol Cell 2011; Andersen et al. Nat. Struct. Mol. Biol. 2013; Meola et al. Mol Cell 2016.

Our laboratory is actively engaged in characterizing RNA decay pathways within mammalian nuclei. These efforts involve the identification of new pathways and components as well as their substrates. To this end, we use state-of-the-art affinity capture / mass spectrometry approaches and high throughput transcriptome-wide methodologies. However, decay of RNA is by no means just a ‘clean-up-act’ to remove worn out molecules. In addition to its essential role in the quality control of genome expression, RNA turnover is also at the core of gene expression regulation – forming intricate connections to RNA productive systems – thus, being centrally placed to determine RNA/RNP fate. Therefore, we also work towards establishing models for how regulated RNA turnover helps control key biological processes. To this end, we are mainly using mouse embryonic stem cells and their ability to differentiate and elicit various gene expression programs.

Pervasive transcription

Our work on pervasive transcription has for example revealed that gene promoters are inherently bidirectional. The RNA produced from a gene is usually protected from exosome attack, whereas the so-called PROMoter uPstream Transcripts (PROMPTs) produced in the opposite direction contain poly (A) sites and other transcription terminators close to their transcription start sites. This triggers RNA decay by the exosome. Our laboratory was the first to identify PROMPTs and has since contributed to the identification of numerous mammalian nuclear RNA exosome targets. For further reading see: Preker et al. Science 2008; Ntini et al. Nat. Strict. Mol. Biol. 2013; Andersson et al. Nat. Comm. 2014; Chen et al. Nat. Genet. 2016; Iasillo et al. Nucleic Acids Res. 2017.

Genomes are pervasively transcribed. Our laboratory aims to identify physiologically relevant transcription events from the sea of spurious events that constantly produce unstable transcripts. This includes new aspects of transcription interference and/or activation as well as the putative identification of new physiologically meaningful RNAs. Such uncovering of new gene regulatory concepts created by pervasive transcription is of interest to us in both the ’normal’ situation and in cases where cells are perturbed by diseases such as cancer or neurobiological defects.

Nonsense-Mediated Decay

snoRNA host genes are highly transcribed thereby producing large amounts of pre-mRNAs that are processed to spliced mRNAs and snoRNAs. snoRNA host genes are more prone to produce NMD-susceptible mRNAs than normal protein-coding genes. One interpretation of this is that snoRNA host genes can switch between the production of protein-coding mRNA and non-coding NMD-susceptible RNA by e.g. alternative splicing to adjust protein expression levels while maintaining an unaltered high level of snoRNA production.?

Our laboratory is studying the cytoplasmic RNA degradation pathway nonsense-mediated mRNA decay in human cells (NMD; Lykke-Andersen and Jensen, 2015). We are interested in both mechanistic aspects of NMD as well as its role in the general control of eukaryotic gene expression. In particular, we are studying the function of the endonuclease SMG6 and its interplay with other nucleases in NMD (Eberle et al., 2009; Lykke-Andersen et al., 2014). Additionally, we have recently taken an interest in the relationship between NMD and snoRNA host genes. These highly expressed genes encode mRNAs or mRNA-like transcripts from their exons and snoRNAs from one or more introns. Because maturation of intron-hosted snoRNAs generally depends on the splicing process, the spliced RNA can be considered as a by-product of snoRNA production. In many cases this ‘by-product’ acts as a normal mRNA and encodes functional protein. However, compared to general protein-coding genes, snoRNA host genes have a much more pronounced tendency to give rise to NMD-susceptible mRNAs (Lykke-Andersen et al., 2014). There are several possible explanations for this, which we are actively investigating. In combination, our studies of the mechanism and global impact of NMD may reveal novel insights about the regulation of eukaryotic gene expression.

RNA Synthesis and Decay in S. cerevisiae

Model based on (Schmid et al., Cell Reports 2015), depicting how the nuclear poly(A) binding protein Nab2p impacts mRNA production. In the presence of Nab2p in the nucleus, the protein binds and protects mRNAs, which can then get exported to the cytoplasm for translation (left panel). mRNAs that are not protected by Nab2p get degraded in the nucleus by the exonucleases Rrp6p and Dis3p (right panel).

In our lab, we use the budding yeast Saccharomyces cerevisiae to study RNA metabolism in the cell nucleus and focus on the relationships between RNA synthesis, decay and export. To identify relevant substrates of key players, we use rapid nuclear protein depletion schemes (‘anchor-away’ or ’degron’ approaches), coupled with genome-wide methodologies for measuring transcription and RNA synthesis as well as RNA fluorescense in-situ hybridization. Nuclear RNA metabolism is not only an intricate component of the gene expression machinery but also provides opportunities for regulation of transcript levels and for establishing cellular RNA homeostasis via functional coupling to transcription and cytoplasmic RNA decay. Identification of such principles is the primary target of our yeast work. Indeed, we have previously identified new paradigms for gene expression control based on polyadenylation, poly(A) binding proteins, nuclear mRNA export and RNA 3’-5’ exonucleolytic decay (Saguez et al. 2008; Schmid et al. 2012; Schmid et al. 2015; Schmid et al. 2018; Tudek et al. 2018). Consequently, our yeast work provides important insights in its own right, but also contributes important inspiration for projects in mammalian cells.