Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - SPLICING IN MITOSIS (Regulation of cell division by alternative splicing)

- Summary description of the project objectives

In this report, we summarize the research activity performed by Dr. Robert Weatheritt during the outgoing phase of the Marie-Curie IOF at the Donnelly Centre, University of Toronto under the supervision of Professor Benjamin Blencowe.

The main goals of this multidisciplinary project are:
1. mRNA sequencing experiment to determine transcriptomes of HeLa cells
2. further development of computational pipeline for the analysis of mRNA-Seq data and analysis of data
3. development of computational pipeline to analyse proteomic datasets using a mRNA-Seq customizable splice junction search database
4. investigate splice variants for differences in stability and sub-cellular localisation using SNAP- based microscopy

5. investigating how alternative splicing alters the protein stability of splice variants
6. investigating how alternative splicing alters the protein stability of splice variants via changes in post-transcriptional and translational control

7. the creation of a web-based resource to allow research community access to data

The seven aims above correspond to the tasks to be accomplished in 36 months. In the proposal’s timeline, we planned to perform 5 of the 7 aims during the outgoing phase (24 months). According to this we have completed the first 3 tasks, as well as parts of aims 4, 5 and 6. The remaining part 7 will be performed during the returning phase at the Laboratory of Molecular Biology (LMB) in Cambridge.

- Description of the work performed since the beginning of the project

During the first months of the project Dr. Weatheritt began the task of synchronizing cells with chemical blocks in order to undertake next-generation sequencing on these samples. However, soon after beginning the Marie Curie fellowship a high-quality sequencing dataset, which followed this procedure, became available. A collaboration was therefore established with the group at the University of North Carolina that produced this dataset. While this collaboration was being established, the fellow collaborated in developing a computational pipeline to analyse RNA-Seq data for all types of alternative splicing events. The development of this pipeline and the collaboration enabled the identification of a wide range of periodic alternative splicing events at multiple time-points during the cell cycle. In the second phrase of this project, Dr Weatheritt extended the computational pipeline to also analyse ribosome-profiling data for alternative splicing events. This enabled the fellow to identify stable splice variants that are engaged by the ribosome, and likely translated, in the cell cycle stage dependent manner.

- Description of the main results achieved so far

The successful completion of the first two stages of the proposal by Dr. Weatheritt resulted in the identification of ~1,300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels.
Many of
the periodically spliced genes are controlled by the SR protein kinase CLK1, whose protein levels undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control.

The completion of adapted versions of aim 3 and aim 6 facilitated the fellow to identify that at least 75% of human exon skipping events detected in medium to high abundance transcripts using RNA-Seq data are also detected by ribosome profiling data. Dr. Weatheritt further identified that relatively small subsets of functionally related splice variants are engaged by ribosomes at levels that do not reflect their absolute abundance, indicating an important role for AS in modulating translational output. This mode of regulation is associated with the control of the mammalian cell cycle. These results suggest that a major fraction of splice variants is translated, and that specific cellular functions including cell cycle control are subject to AS-dependent modulation of translation output.

- Expected final results and their potential impact

The successful completion of the outgoing phase of the project resulted in two publications that partially fulfil the major tasks of the project. The expected work in the final stage of the project will be further publications of the approaches and methods used in this project.

This project has identified for the first time the importance of alternative splicing in cell cycle regulation. Together with the recent identification that splicing factors have high mutation rates in cancer suggests alternative-splicing regulation may be a novel target for therapeutic intervention in the treatment of cancer.

Reported by

United Kingdom


Life Sciences
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