Splicing surprise for yeast researchers
A novel pathway for mRNA processing in the cell is important for fine-tuning gene expression, researchers at Oxford and Edinburgh have discovered.
Dr Monica Passoni and Adam Volanakis together with Dr Cornelia Kilchert and Sneha Shah in Dr Lidia Vasiljeva’s lab in the department, collaborating with Dr Sander Granneman and Dr Ralph Hector at the University of Edinburgh, have described the finding in a paper in Genes and Development (1).
They show how the new pathway involving mRNA splicing can function to control expression of genes in budding yeast in a very different way from that of conventional splicing.
Model describing SMD and conventional splicing. On the left, the pre-mRNA undergoes splicing to produce mRNA that is translated into protein in the cytoplasm. In conventional splicing, splicing is required for production of functional mRNA and protein. However, if SMD occurs, the splicing products are degraded by the nuclear exosome leading to down-regulation of mRNA levels (Click to enlarge)
In eukaryotic cells, many mRNAs once transcribed must be processed in the nucleus to become functional. Splicing of mRNA is one of these processing steps. Through a series of reactions, the introns are removed from the pre-mRNA leaving mature mRNA which is exported to the cytoplasm to be translated into protein.
In yeast, most genes are non-intronic and are not thought to be spliced. But Dr Vasiljeva’s recent work suggests that this may not be quite the case.
She and her colleagues have found that splicing of the transcripts from some of these non-intronic genes does occur, but the outcome is very different from conventional splicing. The process results in unstable RNA products that are degraded in the nucleus, never making their way out to the cytoplasm. The degradation has a functional role in regulating gene expression making this work the first description of such a role for the splicing machinery.
The group came across the finding by purifying the spliceosome, the machinery that carries out splicing. When they analysed the RNAs bound to the spliceosome, in collaboration with the Granneman lab, they were surprised to find the transcripts of many non-intronic genes.
To see how general the phenomenon is, they looked genome-wide at the impact of mutating the spliceosome. For the majority of conventionally spliced genes, they found that the mRNA levels decreased – as expected because splicing is required to produce functional mRNA. But the mRNA levels for a significant proportion of non-intronic genes increased.
They followed the RNA of one particularly interesting non-intronic gene, BDF2. ‘The gene was one of the most prominent hits in the RNA analysis,’ explains Dr Vasiljeva. ‘It codes for a protein involved in transcription initiation and chromatin remodelling, and its expression levels are known to be regulated by different stress conditions.’ The level of Bdf2 is also coordinately regulated with Bdf1, a gene with similar functions.
The group’s analysis confirmed that the BDF2 transcript did undergo splicing which resulted in degradation of the RNA by the nuclear exosome – a process they term spliceosome-mediated decay (SMD). In addition, they found that this degradation of BDF2 is dependent on BDF1, providing a plausible explanation of how expression of the two genes is regulated.
Lidia Vasiljeva (second from right) and her group in the department
Having identified another splicing pathway with a very different outcome from conventional splicing, the group is intrigued to know more about the process. One immediate question is what directs transcripts down one particular splicing pathway over the other?
Dr Vasiljeva has ideas about how this might happen. ‘We speculate that perhaps this could be explained by a difference in kinetics. If the kinetics of splicing is slow, it could allow the RNA degrading machinery in the nucleus to attack the RNA.’ In contrast, conventional splicing is likely to happen quite efficiently – as soon as RNA is spliced, export factors assemble onto the mature mRNA and transport it to the cytoplasm.
For the genes that have been considered as non-intronic up to now, she suggests that the splicing products may not have been detectable because the spliced RNAs are degraded so quickly. She also thinks that the splicing elements which are required to make the process happen, may be sub-optimal and therefore unable to promote efficient splicing.
These are hypotheses which she is looking forward to testing, as well as trying to answer many other questions about how the newly-discovered splicing process occurs – for example, what spliceosome complex is used and how the exosome is recruited.
‘We need to find out what the mechanism is that specifies this pathway,’ says Dr Vasiljeva. ‘How is specificity of the exosome achieved and what does it recognise? The machinery is targeting specific, fully spliced RNAs as well as splicing intermediates for degradation.’
Beyond understanding the mechanistic details, there is the wider biological significance of SMD to address. Researchers are intrigued to know whether the process happens in higher eukaryotes and what its role there might be. Dr Vasiljeva points to a phenomenon in which gene expression is regulated via alternative splicing, that plays a very important role during development.
‘In more complex organisms, alternative splicing can negatively regulate gene expression,’ she comments, but adds that whilst this involves the RNA decay machinery, the mechanism is quite different from SMD. It is early days yet for SMD, but Dr Vasiljeva is keen to start exploring its potential importance.