ECRs at ICaMB: RNA Quality Control


Claudia SchneiderIn the latest of our series focussing on the ECRs in ICaMB, we feature Dr Claudia Schneider. Claudia obtained her PhD from the Philipps-University in Marburg, Germany. She then moved to the UK to work with Prof David Tollervey at the Wellcome Trust Centre for Cell Biology in Edinburgh. In 2011, she was awarded a Royal Society University Research Fellowship and started her own lab at ICaMB. Here, Claudia describes her research, and how being alarmed during her postgraduate studies triggered her long-term research interest.

By Dr Claudia Schneider

Hi, my name is Claudia Schneider, and my Royal Society University Research Fellowship has allowed me to set up my own group here at ICaMB to study enzymes involved in RNA processing and quality control.

I am originally from Germany, where I did my undergraduate studies and my PhD. Many people might think that Germany is the land of cars or lederhosen – but in truth it is really the land of bread (and beer!). It might therefore not come as a surprise that baker’s yeast has become my favourite model organism.

Click on the image to find out how to make these budding buns!

Click on the image to find out how to make these budding buns!

During my undergraduate studies I was first introduced to RNA and I was (and am still) amazed by its many known and still emerging functions in the cell. We now know that almost the entire eukaryotic genome is transcribed, but only a small fraction of the transcripts are protein-coding messenger RNAs (mRNAs). The others are stable and unstable non-coding RNAs (ncRNAs), which are involved in all aspects of gene expression. RNA molecules are often extensively processed before they’re functional, and each processing step is subject to quality control mechanisms. If you want to know more about the life and death of non-coding RNAs, have a look at this recent review.

Yeast has not always been my first choice to study RNA metabolism, since the object of my PhD project in Prof Reinhard Lührmann’s lab turned out to be completely missing in baker’s yeast. Back then I worked on nuclear pre-mRNA splicing, the removal of non-coding introns from precursor mRNAs catalysed by the spliceosome. It was an exciting time in the splicing field: A second low abundance “minor” spliceosome had just been discovered in most multicellular eukaryotes (with the strange and still not readily explainable exception of C. elegans), and this complex is not present in yeast. The minor spliceosome recognises a rare class of introns (<0.5%) with different consensus sequences at the splice sites and has since been linked to a number of human diseases. During my PhD, I purified and biochemically characterised the snRNP components of this unusual pre-mRNA splicing machinery in human and Drosophila cells.

Since then, I have been fascinated by biochemical and enzymatic assays involving RNA such as in vitro splicing assays, where in vitro transcribed pre-mRNAs are mixed with purified spliceosomes. The goal of such an experiment is to observe precise and (hopefully) pretty intron removal in the test tube – but, to my great annoyance, success was every so often hampered by a powerful ribonuclease (RNase) contamination in the assay that completely trashed the precious RNA substrates. Generic and aggressive RNases like RNase A are found on our skin, are incredibly stable and can even survive boiling.

Common decoration on lab surfaces during my PhD

Common decoration on lab surfaces during my PhD

It is therefore fair to say that my scientific career was majorly influenced by constant warnings by my mentor, who told me that all RNases are evil and must be destroyed. However, for my postdoc, I decided to face my fears and look these evil RNases in the eye, in the humble model system yeast. During my time with Prof David Tollervey at the University of Edinburgh, I learned that there are many different types of RNases, and only very few of them are promiscuous and chop RNA to bits.

The majority of RNases are very sophisticated and versatile enzymes. Several RNases are capable of degrading only specific RNA molecules, or only function under certain circumstances, and protein co-factors often assist in substrate recognition. RNases are crucial elements in RNA quality control or surveillance systems, which distinguish aberrant from “normal” RNA molecules. One clinically important RNA surveillance pathway is called “nonsense-mediated decay” or NMD, and this system recognises and degrades a specific class of defective mRNAs to limit the synthesis of truncated and potentially toxic proteins. NMD defects are linked to ~30% of all inherited human diseases (e.g. Duchenne muscular dystrophy and forms of b-thalassemia) as well as certain types of cancer. In addition to quality control/surveillance, where RNAs are mostly completely degraded, a growing number of RNases have been shown to be responsible for precise processing or “trimming” of precursor RNA molecules to produce their functional forms.

Exonucleases were long believed to be the main players involved in RNA recognition and processing/turnover. However, this model was recently challenged by the identification of endonucleases containing PIN (PilT N-terminus) domains, which appear to play key roles in RNA metabolism. Eight PIN domain proteins and therefore putative endonucleases are encoded in the genome of budding yeast, and this includes three largely uncharacterised “orphan” nucleases.

Overall it is still puzzling to me how individual RNases “make the decision” to either completely degrade or carefully process a specific RNA. Given the ever-growing number of non-coding transcripts in the cell, I am also keen to know which RNases are responsible for which substrates and what the so-far uncharacterised putative PIN domain endonucleases in yeast are doing!

To this end, our lab is using an RNA-protein cross-linking method called “CRAC” (UV cross-linking and analysis of cDNA) to identify the targets of PIN domain endonucleases on a transcriptome-wide scale. The CRAC method and the machinery to cross-link yeast cultures were developed by Sander Granneman at the University of Edinburgh, when we were both PostDocs in Prof David Tollervey’s lab. Sander now has his own lab too, and he runs a CRAC-blog. Interestingly, the cross-linking device we are using was originally designed to sterilise sewage water, but it is now also commercially available for research. With this setup, yeast cells are cross-linked while they are growing in culture, which is crucial to identify the often very transient interactions between nucleases and their target RNAs. This system provides a huge advantage over more traditional cross-linkers like the “Stratalinker”, which requires pelleting and cooling the cells on ice before cross-linking. It is, however, also much bigger and takes up a whole bench in the lab – but I guess there is a drawback to everything! In any case: if you want to find RNA targets for your favourite yeast protein – get in touch!!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Transcriptome-wide RNA-protein interaction analyses generate huge datasets and we use RNA binding and nuclease assays, as well as co-precipitation studies, to validate the in vivo cross-linking results for individual PIN domain endonucleases.

With the help of an ERASMUS exchange student, Franziska Weichmann, who spent 6 months in my lab last year, we have made good progress with two putative PIN domain endonucleases that are linked to ribosome biogenesis. We were able to identify their binding sites on the pre-ribosomal RNAs, as well as co-factors that are important to recruit them into the pre-ribosome. We have also set up an in vitro system with recombinant proteins, and we are currently trying to convince one of them to specifically cleave its proposed rRNA substrate in the test tube – and we are slowly getting there…..

Like the other ICaMB ECRs, who posted on this Blog before, I would like to finish by saying that having my own lab has been an exciting and (on most days) enjoyable adventure so far – and I am looking forward to the next set of challenges…

ECRs at ICaMB: Copying the blueprint of life – Understanding DNA replication


Heath MurrayIn the latest of our series focussing on the Early Career Researchers (ECRs) in ICaMB, we feature Dr Heath Murray.  After completing undergraduate studies at the University of California, Los Angeles and then obtaining his Ph.D. from the University of Wisconsin-Madison, Heath came to the UK to join the lab of Prof Jeff Errington in Oxford. From there he re-located to ICaMB, and in 2009 was awarded a Royal Society University Research Fellowship. Here, Heath describes his research into the mechanisms of DNA replication, and explains why he became interested in this field.

By Dr Heath Murray

Hello, my name is Heath Murray and I’m a Royal Society University Research Fellow in ICaMB’s Centre for Bacterial Cell Biology (CBCB) studying DNA replication. DNA is one of the most important molecules required for life because it encodes the information, or the blueprint, used to build a cell (i.e. the most basic unit of an organism). In order for a cell to create new cells it must synthesize an exact copy of its DNA, an extraordinary process when you consider that the genomes of most cells contain millions of individual DNA subunits!

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacteria are ideal model systems to study this fundamental process because they are much less complex than human cells (e.g. all of their DNA is encoded by a single chromosome, whereas humans have 23), and this allows us to understand how they work at the greatest possible level of detail.

I was introduced to bacteria when I was an undergraduate student and the effect was transformative. My mentors taught me how to add a specific gene (a DNA sequence) to a bacterial cell, and if it worked properly then the bacteria would turn blue!

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

That basic genetic experiment was one of the coolest things I had ever done, and from that point on I worked hard to learn the trade of “bacterial genetics”.

Today my research group focuses on understanding how DNA replication is controlled so that each new cell will end up containing an exact copy of the genetic material from its predecessor. We employ a wide range of complementary experimental techniques: genetic engineering of bacterial strains, biochemical analysis of purified proteins, and fluorescence microscopy.

In the hot room to check my plates.... I haven't even stopped to take my jacket or backpack off yet!

In the hot room to check my plates…. I haven’t even stopped to take my jacket or backpack off yet!

Fluorescence microscopy is a particular strength of the CBCB because there are several bespoke systems specifically designed for bacterial cells (bacteria are 10-100 times smaller than most human cells). One of the core approaches we use is to genetically engineer a protein we want to study so that it will be fused to a special reporter protein called GFP (Green Fluorescent Protein, originally isolated from jellyfish!) within the cell. Using this approach we can then visualize where our test protein is because it fluoresces when exposed to a specific wavelength of light. Some of our microscopes are so sophisticated that we can observe the location of single proteins and track their movements within living cells.

At the bench.

At the bench.

One of the approaches we often use is to visualize specific regions of the genome within living cells. First, a specific DNA binding protein ( called “LacI”) is genetically fused to GFP. Second, the DNA sequence recognized by LacI (called “lacO”) can be genetically integrated into any location of the genome. Since I study DNA replication, I am particularly interested in the site of the bacterial chromosome where DNA synthesis is initiated (called the “replication origin”). Third, fluorescent dyes are added to cells that bind to the cell membrane and the DNA. Finally, we utilize our fluorescent microscopes to visualize the location of replication origins within individual cells. In the image shown, the live bacterial cells contain chromosomes that are in the process of being replicated, and therefore they have duplicated and separated their replication origins!! This image also emphasizes the fact that although bacteria lack the organelles found in eukaryotic cells, they are nonetheless highly organized (notice how the replication origins are characteristically located at the outer edge of each chromosome).

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

Well that’s it for my first ICaMB blog! I hope you enjoyed hearing about how I became interested in bacterial genetics and about my work on bacterial DNA replication. Please feel welcome to contact me if you have any questions or if you would like further information regarding my research.



Royal Society URF

Centre for Bacterial Cell Biology (CBCB)


ECRs at ICaMB: For he’s a jolly good (RS) Fellow

As you saw from our post a couple of weeks ago, ICaMB has recruited several new PIs. To make sure we all know more about them and their research, we decided to have a series specially dedicated to them: Early Career Researchers (ERCs) at ICaMB. First in the series is Dr Kevin Waldron.

by Dr Kevin Waldron

As some of you will already know, I was awarded a Sir Henry Dale fellowship in 2012. This is a new funding scheme for early career researchers, which is co-funded by the Wellcome Trust and the Royal Society. The scheme provides exceptional research support for fellows, including funding a postdoctoral research assistant for the duration of the project (5 years initially, with potential extension for a further 3) as well as all direct research costs, with the aim of supporting the researcher while they forge an independent research career and carve out their research niche. As a result they are highly coveted and extremely competitive – the interview at the Wellcome Trust in July 2012 was probably the most intimidating experience of my career. My award was one of the very first round, in which 10 awards were given from a pool of about 100 applications.

Enjoying the sights of Ha Long Bay (Vietnam)… and doing some deep thinking about my science, of course…

Though the grant was awarded in Summer 2012, I only activated the grant in May 2013 (primarily so that I could swan off on an extended holiday travelling round South East Asia – but that’s another story). Since May there have been two new additions to my lab team; Emma Tarrant, a postdoctoral research associate funded by the fellowship grant, and Anna Barwinska-Sendra, a new PhD student. This combination of good funding and great new people will allow us to really get our teeth into our research question over the coming years, free of the hassle of grant writing and worrying about where the next funds are coming from.


I have been studying bacterial metal homeostasis since I started my PhD with Prof. Nigel Robinson (now in Durham), way back in 2003. During that period I studied cyanobacteria, but my current research is focused on the Gram positive mammalian pathogen Staphylococcus aureus. S. aureus has become a major medical problem in recent decades due to the rise of strains that are resistant to multiple conventional antibiotics, with the term “superbug” and the acronym “MRSA” entering the popular consciousness – see for example here, here and here. The organism is particularly problematic in hospitals, where it causes significant morbidity and mortality (see for example these UK and US studies). Though infection control measures introduced in hospitals are proving successful in reducing the number of S. aureus-related deaths, both the prevention and treatment of hospital-acquired infections remain major burdens on patients and on the NHS budget. S. aureus is also problematic in farm animals, being a major cause of bovine mastitis and of lameness among broiler chickens.

My favourite metal

One intriguing new approach to prevention of infection in such settings is the use of solid copper or copper-containing alloys. Copper surfaces show wide-spectrum antimicrobial activity and are certified by the US EPA as antibacterial and ‘self-sanitising’. Multiple trials have shown that these materials can reduce bacterial copper load on touch surfaces in hospitals (see here for example), and the effects on disease transmission are currently being tested. In fact, this is not a new approach at all: as this YouTube clip explains, copper has been used since ancient times to sterilise drinking water and to treat minor ailments, even being mentioned by Hippocrates in ~400 BC. Copper is the active ingredient in numerous agricultural fungicides including Bordeaux mixture, in use since 1885.

The mechanisms by which copper, either as metal salts or as solid metal surfaces, kills bacteria are unknown. Dissolved copper ions are thought to play an important role in the killing mechanism even from solid copper surfaces. In fact, the toxicity of excess concentrations of essential metal ions (as opposed to non-essential metals such as lead and mercury) has been historically under-studied and has merely been seen as a confounding factor of studies of normal metal homeostasis. Copper is redox-active, a potent Fenton catalyst, and therefore may catalyse the production of reactive oxygen species in vivo. It is also at the top of the Irving-Williams series, meaning it will bind extremely tightly to proteins, potentially having deleterious effects on their function when present in excess.

Our goal is to identify and understand these toxicity mechanisms, both to shed light on new aspects of metal homeostasis and to find better ways of exploiting this toxicity for medical and commercial applications. To do this we’re going to use a combination of multiple experimental approaches in collaboration with a number of world experts in diverse fields. We will use proteomic methods to identify S. aureus proteins that become aberrantly associated with copper under high-copper growth conditions, and then characterise the functional effects of those aberrant associations using traditional biochemistry and molecular genetics. We will combine this with metabolomics studies of the effects of copper toxicity on bacterial metabolism. Finally, we will use genetic screens to identify mutant strains that display increased copper resistance. Together, this should give us a comprehensive view of the effects of copper toxicity on the cell, with the aim of elucidating the multiple mechanisms by which excess copper interferes with normal function.

The Waldron lab group: Jack Stevenson, Emma Tarrant, Anna Barwinska-Sendra, Stuart York and Kevin Waldron (left to right)


So we’re just starting off on a long-term quest, with lots of experiments to keep us busy for the next few years. It’s an exciting time in the Waldron lab.






Sir Henry Dale Fellowships
Wellcome Trust
Royal Society