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Recent Research

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.

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.

New approaches for new questions

This is a very exciting period in RNA biology; numerous novel RNA species, RNA functions and RNA targets have recently been discovered, and it seems very likely that many remain to be identified. Most RNAs function within ribonucleoprotein (RNP) complexes, via base-pairing with target RNAs. However, both RNA folding and intermolecular RNA base-pairing remain difficult to reliably determine experimentally or using bioinformatics. We previously developed a protein-RNA crosslinking technique termed crosslinking and analysis of cDNA (CRAC). In this, a tagged “bait” protein is UV-crosslinked to associated RNAs in vivo, and then purified under denaturing conditions. RNA fragments are recovered and the precise protein binding sites are identified by deep sequencing of cDNAs. To identify RNA-RNA interactions, we developed the related technique of crosslinking, ligation and sequencing of hybrids (CLASH). During CLASH analyses, the two strands present in base-paired, RNA-RNA duplexes that associate with the bait protein are ligated together and then recovered as a chimeric cDNA.

Applying this appraoch to human microRNAs (miRNAse) generated data sets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, nonseed base pairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA base pairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.

Distinguishing lncRNAs and mRNAs

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 (lncRNA) transcripts are so common in budding yeast and human cells that almost the entire genome is transcribed by RNA polymerase II. This suggested that lncRNAs might also play important roles in establishing and modifying chromatin structure.

LncRNAs and mRNAs are both transcribed by Pol II and acquire 5’ caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3’ end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3’ cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of “lncRNA-like” mRNAs regulated by the nuclear surveillance machinery.

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.

The CRAC UV crosslinking technique identified numerous pre-rRNA binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30 and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to the sequence flanking A3 and to the RNA component of RNase MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural protein factors and NTPases. A key role in coordinating pre-ribosomal assembly and processing was confirmed by “Miller” chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and pre-ribosome compaction greatly reduced.