Co-workers:

Our aim is to understand the nuclear pathways that process newly transcribed RNAs and assemble RNAprotein complexes, the mechanisms that regulate these pathways and the surveillance activities that monitor their fidelity. We are addressing these questions using a combination of systems biology, genetics, biochemistry and cell biology approaches in the budding yeast Saccharomyces cerevisiae.
To better understand both RNA maturation and surveillance, we developed techniques for quantitative analysis supported by mathematical modelling (Kos and Tollervey, 2010) and for UV cross-linking and analysis of cDNAs (CRAC) to precisely identify sites of RNA-protein interaction (Granneman et al., 2009). These are having a dramatic effect on our understanding of ribosome synthesis (Granneman et al., 2010; van Nues et al., 2011). In budding yeast long, non-protein coding RNA (ncRNA) transcripts are common. CRAC analyses of the in vivo targets for nuclear RNA surveillance factors (Wlotzka et al., 2011) identified some ~642 long antisense ncRNAs (asRNAs) and ~178 long, intergenic noncoding RNAs. These are predicted to alter chromatin structure, aiding the optimization of gene expression patterns in a changing environment. Surveillance targets were enriched for non-encoded A-rich tails, which can act as single-stranded “landing pads” for
RNA degradation by the exosome complex. The tails on surveillance targets were generally very short (A1-5), potentially explaining why these RNAs are destabilised, whereas mRNAs are stabilized by longer poly(A) tails. Unexpectedly, RNA polymerase III transcripts formed a major class of substrates identified for the nuclear RNA surveillance system (Figure 1).
 
We also reported that transcription pausing and r-loop formation are frequent during pre-rRNA transcription (El Hage et al., 2010; French et al., 2011). RNA Polymerase movement during transcription forces the rDNA to rotate. This builds up transient positive torsion ahead of Pol I, resisting strand opening and pausing transcription elongation. In contrast, negative torsion behind the polymerase facilitates DNA strand opening, permitting R-loop formation with the nascent pre-rRNA. These R-loops are cleaved by RNase H, releasing truncated pre-rRNA fragments that are degraded by the TRAMP/ exosome RNA surveillance system.
Finally, the identification of RNA-RNA interactions is essential for the detailed understanding of many biological processes. We have described a highthroughput method to experimentally identify intramolecular and intermolecular RNA-RNA interactions by crosslinking, ligation and sequencing of hybrids
(CLASH) (Figure 2) (Kudla et al., 2011). The CLASH approach should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.

Selected publications:

Wlotzka, W., Kudla, G., Granneman, S., Tollervey, D. (2011) The nuclear RNA polymerase II surveillance system targets polymerase III transcripts. EMBO J. 30, 1790-1803.

Kudla, G., Granneman, S., Hahn, D., Beggs, J.D. and David Tollervey, D. (2011) Crosslinking, ligation and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc. Natl. Acad. Sci. USA. 108, 10010-10015.

El Hage, A., French, S.L., Beyer A.L. and Tollervey, D. (2010) Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev. 24, 1546-58.