Lab Members:

Major questions and approaches

The research of the lab addresses key questions in nuclear RNA metabolism. The research can be divided into two broad areas - ribosome synthesis and nuclear RNA surveillance. The individual projects are inter-linked: 1) Thematically, because eukaryotic cells use a common set of enzymes and cofactors to perform both processing and degradation of diverse RNA species. 2) Methodologically, because we use a set of related techniques to uncover the maturation, surveillance and function of different classes of RNA.

RNA Surveillance:

Eukaryotic cells contain a huge range RNA species, almost all of which are synthesised by post-transcriptional processing. My group is analysing the mechanisms and regulation of RNA processing and turnover. This description sounds very general - and with good reason. We have a long-standing interest in ribosome synthesis and, starting from the analysis of pre-rRNA processing components, we have found that both the synthesis and degradation of many different types of RNA depends on a set of common nucleases and cofactors. These are recruited in different combinations to many different substrates.

The degradation of nuclear pre-mRNAs and cytoplasmic mRNAs, as well as accurate 3' processing of many stable RNA species, involves the exosome - a complex of ten core proteins with 3' to 5' exonuclease activity. Since the exosome mediates both precise RNA processing and total RNA degradation (in some cases of the same RNA species under different conditions) the regulation of its activity is of key importance and is mediated by multiple nuclear and cytoplasmic cofactors.

Functions of non-coding RNAs in the yeast nucleus:

Budding yeast lacks the siRNA system that plays an important role in heterochromatin dynamics in many other Eukaryotes. In contrast, recent studies indicate that long non-protein coding (ncRNA) transcripts are so common in budding yeast and human cells that almost the entire genome is transcribed by RNA polymerase II. This suggested that long ncRNAs might also play important roles in establishing and modifying chromatin structure.
    We have shown that heterochomatic regions of the S.cerevisiae genome, the telomeres and rDNA spacers, are actively transcribed, but the resulting ncRNA products are targeted for very rapid degradation by the exosome complex. Remarkably, at least 4 different exosome cofactors participate in the degradation of individual ncRNA species, the TRAMP polyadenylation complex, the Nrd1/Nab3 complex, and the RNA-binding proteins Rrp47 and Mpp6. We predict that this functional redundancy is a key feature of the very rapid degradation that maintains ncRNA transcripts at low levels.
    ncRNAs also function in the euchromatic regions of the yeast genome. The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar concentrations. However, we observed that under repressive conditions it contains trimethylated histone H3 K4, indicative of active transcription. Binding of the transcription factor Reb1 in the cluster initiates transcription of a long ncRNA that is expressed reciprocally to the GAL1 and GAL10 mRNAs. Levels of the ncRNA are regulated by degradation involving the TRAMP/exosome system. At steady-state less than 10% of yeast cells contain a GAL ncRNA molecule. Production of the ncRNA causes multiple, repressive chromatin modifications across the GAL gene cluster, including histone H3 K14/18 and K27 deacetylation and trimethylation of H3 K36.

Ribosome synthesis:

The synthesis of  ribosomes is a major metabolic activity in any dividing cell, and is closely linked to growth control. Despite a great deal of work, there remain many unanswered questions about the ribosome synthesis pathway, even in budding yeast where it is best understood.

In our current analyses of ribosome synthesis we have been applying techniques of systems biology:

High resolution kinetic analyses combined with mathematical modeling.

Quantitative analyses of the binding and release of the 75 small nucleolar RNAs that directly participate in ribosomal RNA processing and modification.

Systematic identification of the binding sites for ribosome synthesis factors - using a newly developed method for RNA-protein cross-linking and cloning of the bound RNAs (CRAC).

Each of these novel approaches is giving fresh insights into the highly complex ribosome synthesis pathway.