Beggs, Jean

Granneman, Sander

Barrass, James David

2021-07-22T12:41:05Z

2021-07-22T12:41:05Z

2021-07-31

Over the course of 10 years and 14 publications, 7 of which are described in this thesis, I made significant contributions to the field of RNA processing, particularly RNA splicing in budding yeast. These contributions have been both in knowledge accrued and techniques developed and optimised. For the Ribo1 reporter (chapter 2), I developed the RT-qPCR assays, RNA copy number/cell estimation and 3’ end cleavage assay. These show that the minimum time taken for signal induction, recruitment, assembly of transcription factors and ultimately transcription, is in the order of 4 minutes, with transcription rates of 60 to 90 seconds per kb. The 3’ end cleavage assay reveals that the initial pulse of transcripts may be spliced only partially co-transcriptionally; presumably splicing factors are recruited to the site of transcription during this period. After this initial phase, in the reporter at least, splicing is almost exclusively co-transcriptional. Mutant transcripts on which spliceosomes assemble but are unable to complete splicing, are targeted for very rapid degradation. I have optimised the thio-labelling technique to the point where I can detect thiolated RNAs just 15 seconds after addition of the 4-thiouracil nucleotide analogue to cell cultures (chapter 3). From my data and transcriptome sequencing (chapter 4), I have constructed models of transcription and splicing, which indicate that co-transcriptional splicing is a general feature of most yeast pre-mRNA transcripts and almost all ribosomal protein gene transcripts. Intronic features that act against co-transcriptional splicing include runs of uracils and secondary structure, especially over the branch point adenosine (chapter 4). I have provided a highly detailed protocol and video for thio-labelling of RNA in vivo and its purification (chapter 3). Similarly, I have developed and prepared a comprehensive protocol for auxin-induced protein depletion, to the point where this is the most flexible and least metabolically perturbing technique for doing this in yeast (chapter 6). Applying this method, whilst thio-labelling, I revealed that depleting splicing factors sequesters spliceosome components, rapidly resulting in the cessation of splicing (chapter 7). My microarray results indicate that this is a common consequence of splicing factor deactivation for many or all transcripts. In this thesis I examine RNA metabolism, noting that the processes of transcription, splicing, 3’ end formation and degradation are coordinated and harmonised to optimise fidelity, flexibility and efficiency.

https://hdl.handle.net/1842/37782

http://dx.doi.org/10.7488/era/1058

en

The University of Edinburgh

Alexander, R. D., Barrass, J.D., Dichtl, B., Kos, M., Obtulowicz, T., Robert, M.-C., Koper, M., Karkusiewicz, I., Mariconti, L., Tollervey, D., Dichtl, B., Kufel, J., Bertrand, E., Beggs, J.D., 2010. RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3’-end processing in Saccharomyces cerevisiae. RNA 16, 2570–2580

Barrass, J.D., Beggs, J.D., 2019. Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU). JoVE (Journal of Visualized Experiments) e59952.

Barrass, J.D., Reid, J.E., Huang, Y., Hector, R.D., Sanguinetti, G., Beggs, J.D., Granneman, S., 2015. Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling. Genome Biology 16, 282.

Kafasla, P., Barrass, J.D., Thompson, E., Fromont-Racine, M., Jacquier, A., Beggs, J.D., Lewis, J., 2009. Interaction of yeast eIF4G with spliceosome components. RNA Biol 6, 563–574.

Mendoza‐Ochoa, G.I., Barrass, J.D., Terlouw, B.R., Maudlin, I.E., Lucas, S. de, Sani, E., Aslanzadeh, V., Reid, J.A.E., Beggs, J.D., 2018. A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast. Yeast 36 (1) 75-81.

Barrass, J.D., Mendoza-Ochoa, G.I., Maudlin, I.E., Sani, E., Beggs, J.D., 2019. Tuning Degradation to Achieve Specific and Efficient Protein Depletion. JoVE (Journal of Visualized Experiments) e59874

Mendoza-Ochoa, G.I., Barrass, J.D., Maudlin, I.E., Beggs, J.D., 2019. Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo. RNA Biol 16, 1775–1784.

Kennedy, P.G.E., Barrass, J.D., Graham, D.I., Clemens, G.B., 1990. Studies on the pathogenesis of neurological diseases associated with Varicella-Zoster Virus. Neuropathology and Applied Neurobiology 16, 305–316.

Carver, A., Wright, G., Cottom, D., Cooper, J., Dalrymple, M., Temperley, S., Udell, M., Reeves, D., Percy, J., Scott, A., Barrass, D., Gibson, Y., Jeffrey, Y., Samuel, C., Colman, A., Garner, I., 1992. Expression of human α1 antitrypsin in transgenic sheep. Cytotechnology 9, 77–84.

Carver, A.S., Dalrymple, M.A., Wright, G., Cottom, D.S., Reeves, D.B., Gibson, Y.H., Keenan, J.L., Barrass, J.D., Scott, A.R., Colman, A., Garner, I., 1993. Transgenic Livestock as Bioreactors: Stable Expression of Human Alpha-1- Antitrypsin by a Flock of Sheep. Nat Biotech 11, 1263–1270

Barrass, J.D., Beggs, J.D., 2003. Splicing goes global. Trends in Genetics 19, 295–298

Boon, K.-L., Auchynnikava, T., Edwalds-Gilbert, G., Barrass, J.D., Droop, A.P., Dez, C., Beggs, J.D., 2006. Yeast Ntr1/Spp382 Mediates Prp43 Function in Postspliceosomes. Mol. Cell. Biol. 26, 6016–6023

Houalla, R., Devaux, F., Fatica, A., Kufel, J., Barrass, D., Torchet, C., Tollervey, D., 2006. Microarray detection of novel nuclear RNA substrates for the exosome. Yeast 23, 439–454

Boon, K.-L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearn, C.F., Beggs, J.D., 2007. prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nat Struct Mol Biol 14, 1077–1083.

Kershaw, C.J., Barrass, J.D., Beggs, J.D., O’Keefe, R.T., 2009. Mutations in the U5 snRNA result in altered splicing of subsets of pre-mRNAs and reduced stability of Prp8. RNA 15, 1292–1304.

Grainger, R.J., Barrass, J.D., Jacquier, A., Rain, J.-C., Beggs, J.D., 2009. Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome. RNA 15, 2161–2173.

Alexander, Ross D, Innocente, S.A., Barrass, J.D., Beggs, J.D., 2010. Splicing-dependent RNA polymerase pausing in yeast. Mol. Cell 40, 582–593.

Swiatkowska, A., Wlotzka, W., Tuck, A., Barrass, J.D., Beggs, J.D., Tollervey, D., 2012. Kinetic analysis of pre-ribosome structure in vivo. RNA 18, 2187– 2200

Chathoth, K.T., Barrass, J.D., Webb, S., Beggs, J.D., 2014. A Splicing Dependent Transcriptional Checkpoint Associated with Prespliceosome Formation. Molecular Cell 53, 779–790.

Cordin, O., Hahn, D., Alexander, R., Gautam, A., Saveanu, C., Barrass, J.D., Beggs, J.D., 2014. Brr2p carboxy-terminal Sec63 domain modulates Prp16 splicing RNA helicase. Nucleic Acids Res 42, 13897–13910.

Gautam, A., Grainger, R.J., Vilardell, J., Barrass, J.D., Beggs, J.D., 2015. Cwc21p promotes the second step conformation of the spliceosome and modulates 3′ splice site selection. Nucleic Acids Res 43, 3309–3317.

Tudek, A., Schmid, M., Makaras, M., Barrass, J.D., Beggs, J.D., Jensen, T.H., 2018. A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA. Cell Reports 24, 2457-2467.

Alexander, R.D., Innocente, S.A., Barrass, J.D., and Beggs, J.D. (2010a). Splicing-dependent RNA polymerase pausing in yeast. Mol. Cell 40, 582–593

RNA

RNA splicing

RNA processing

gene transcription

Investigations of RNA production and processing in Saccharomyces cerevisiae

Thesis or Dissertation

Doctoral

PhD(P) Doctor of Philosophy by Research Publications