University of Dundee

Professor John Brown

RNA processing and expression
Position: 
Chair of Molecular Plant Sciences
Affiliation: 
Address: 
University of Dundee at JHI, Errol Road, Invergowrie, Dundee
Full Telephone: 
+44 (0) 1382 568777, int ext
Email: 

Research

Gene expression in plants - Alternative splicing (AS)

Figure 1

Alternative splicing (AS) is a major regulator of gene expression increasing protein complexity and regulating transcript levels. We showed that in Arabidopsis over 60% of genes undergo AS. Although AS is likely to be involved in all plant processes including growth, development, plant defence, stress responses etc., we still know little about the genome-wide contribution of AS or how AS of specific genes impacts physiological or developmental processes.

To address such questions we have performed an ultra-deep RNA-sequencing of a high resolution time-course of Arabidopsis plants transferred to the cold. To quantify gene and transcript expression accurately from RNA-seq data and analyse expression/AS across multiple time-points, we developed the Arabidopsis thaliana Reference Transcript Dataset (AtRTD2), a tool for time-series isoform switches and new analysis methods. In response to cold, over 7k genes are differentially expressed at the transcriptional level, around 800 at both transcriptional and AS levels, an 1.6k are regulated only at the AS level (Figure 1). Therefore, around one-third of the genes in Arabidopsis have altered expression/AS in response to cold and nearly 30% of these are regulated by AS; the vast majority are novel cold response genes.

Figure 2

The expression profiles of individual transcripts illustrate changes in AS and that many occur rapidly after onset of cold (Figure 2a). The high temporal resolution of the time-course demonstrates rapid and dynamic AS activity in response to lowering temperature with waves of transcription and AS in the first few hours of cold involving thousands of genes (Figure 2b and c).

We have identified transcription and splicing regulators with the earliest and largest expression/AS changes as candidate regulators of early stages of the cold response. Four such genes have been previously shown to be required for freezing tolerance and acclimation. Therefore, AS is a major contributor to regulating changes in gene expression such that plants can deal with hourly, daily and seasonal changes in their environment. By analysing candidate regulators we aim to develop approaches to improve stress tolerance in crop plants and to this end are translating our experience in RNA-seq and AS analyses to crop species (barley, potato) and have shown significant low temperature-dependent AS in barley genes.

 

Publications

  1. Guo, W., Calixto, C.P.G., Brown, J.W.S., and Zhang, R. (2017) TSIS: an R package to infer alternative splicing isoform switches for time-series data. Bioinformatics. 33, 3308-3310
  2. Guo, W., Calixto, C.P.G., Tzioutziou, N., Lin, P., Waugh, R., Brown, J.W.S., and Zhang, R. (2017) Evaluation and improvement of the regulatory inference for large co-expression networks with limited sample size. BMC Systems Biology. 11, 62
  3. Zhang, R., Calixto, C. P. G., Marquez, Y., Venhuizen, P., Tzioutziou, N. A., Guo, W., Spensley, M., Lewandowska, D., ten Have, S., Frei dit Frey, N., Hirt, H., James, A. B., Nimmo, H. G., Barta, A., Kalyna, M., Brown, J.W.S. (2017) A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing. Nucleic Acids Research.45,5061-5073
  4. Calixto CPG, Simpson CG, Waugh R, Brown JWS (2016) Alternative Splicing of Barley Clock Genes in Response to Low Temperature. PLoS One 11:e0168028.
  5. Brown JWS, Calixto CPG, Zhang R. (2016) High-quality reference transcript datasets hold the key to transcript-specific RNA-sequencing analysis in plants. New Phytol. 213: 525-530.
  6. Simpson CG, Fuller J, Calixto CPG, McNicol J, Booth C, Brown JWS, Staiger D. (2016) Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes. Methods Mol Biol. 1398:119-132. 
  7. Russell, J., Mascher, M., Dawson, I. K., Kyriakidis, S., Calixto, C. P. G., Freund, F., Bayer, M., Milne, I., Marshall-Griffiths, T., Heinen, S., Hofstad, A., Sharma, R., Himmelbach, A., Knauft, M., van Zonneveld, M., Brown, J. W. S., Schmid, K., Kilian, B., Muehlbauer, G. J., Stein, N. and Waugh, R. (2016)  Exome sequencing of geographically diverse barley landraces and wild relatives gives insights into environmental adaptation. Nature Genet. Sep;48(9):1024-30.
  8. Zhang, R., Calixto, C.P.G., Marquez, Y., Venhuizen, P., Tzioutziou, N.A., Guo, W., Spensley, M., Frei dit Frey, N., Hirt, H., James, A.B., Nimmo, H.G., Barta, A.,  Kalyna, M. and Brown, J.W.S. (2016)  AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data. bioRxiv 
  9. Carvalho, R.F., Simpson, C. G., Szakonyi, D., Crozet, P., Barbosa, I. C. R., Brown, J. W. S., Baena-González, E. and Duque, P. (2015) The Arabidopsis SR45 splicing factor, a negative regulator of sugar signaling, modulates Snf1-related protein kinase1 stability. Plant Cell Aug;28(8):1910-25.
  10. Brown, J. W. S., Simpson, C. G., Marquez, Y., Gadd, G. M., Barta, A. and Kalyna, M. (2015) Lost in translation: pitfalls in deciphering plant alternative splicing transcripts. Plant Cell 27, 2083-2087
  11. Schlaen, R.G., Mancini, E., Sanchez, S.E., Santangelo, S.P., Rugnone, M.L., Simpson, C.G., Brown, J.W.S., Zhang, X., Chernomoretz, A. and Yanovsky, M. J. (2015) A splicing factor modulates temperature compensation of circadian rhythms in Arabidopsis. Proc Natl Acad Sci U S A. 112, 9382-9387.
  12. Zhang, R., Calixto, C.P.G., Tzioutziou, N.A., James, A.B., Simpson, C.G., Gou, W., Marquez, Y., Kalyna, M., Patro, R., Eyras, E., Barta, A., Nimmo, H.G. and Brown, J.W.S. (2015) AtRTD - A comprehensive Reference Transcript Dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana. New Phytologist 208, 96-101.
  13. Shin, K. H., Yang, S. H., Lee, J. Y., Lim, C. W., Lee, S. C., Brown, J. W. S. and Kim, S. H. (2015) Alternative splicing of mini-exons in the Arabidopsis leaf rust receptor-like kinase LRK10 genes affects subcellular localisation. Plant Cell Rep. 34, 495-505.
  14. Calixto, C.P.G., Waugh, R. and Brown, J.W.S. (2015) Evolutionary relationships among barley and Arabidopsis core circadian clock and clock-associated genes. J. Mol. Evol. 80:108-19.
  15. Bardou, F., Ariel, F., Simpson, C. G., Romero, N., Laporte, P., Balzergue, S., Brown, J. W. S. and Crespi, M. (2014) Long non-coding RNA modulates alternative splicing regulators in Arabidopsis. Developmental Cell 30, 166–176.
  16. Raczynska, K. D., Stepien, A., Kierzkowski, D., Kalak, M., Bajczyk, M., McNicol, J., Simpson, C. G., Szweykowska-Kulinska, Z., Brown, J. W. S. and Jarmolowski, A. (2014) The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana. Nucleic Acids Research 42, 1224-1244.
  17. Petrillo, E., Godoy Herz, M.A., Simpson, C.G., Fuller, J., Fuchs, A., Brown, J.W.S., Barta, A., Kalyna, M. & Kornblihtt, A.R. 2013.  Regulation of plant alternative splicing by the photosynthesis electron transfer chain.  Science 344, 427-430..
  18. Simpson, C.G., Lewandowska, D., Liney, M., Davidson, D., Chapman, S., Fuller, J., McNicol, J.W., Shaw, P.J. & Brown, J.W.S. 2013.  Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and can affect alternative splicing of endogenous genes differentially.  New Phytologist 203:424-436..
  19. Staiger, D. & Brown, J.W.S. 2013.  Alternative splicing at the intersection of biological timing, development and stress responses (Review).  The Plant Cell 25, 3640-56..
  20. Streitner, C., Simpson, C.G., Shaw, P., Danisman, S., Brown, J.W.S. & Staiger, D. 2013.  Small changes in ambient temperature affect alternative splicing in Arabidopsis thaliana.  Plant Signaling & Behavior 8, e24638.
  21. Lewandowska D, ten Have S, Hodge K, Tillemans V, Lamond AI, et al. (2013) Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics. PLoS ONE 8(8): e72207.
  22. James, A., Syed, N.H., Bordage, S., Marshall, J., Nimmo, G.A., Jenkins, G.I., Herzyk, P., Brown, J.W.S. & Nimmo, H.G. 2012.  Alternative splicing mediates responses of the Arabidopsis thaliana circadian clock to temperature changes.  The Plant Cell 24, 961-981.
  23. James, A.B., Syed, N.H., Brown, J.W.S. & Nimmo, H.G. 2012.  Thermoplasticity in the plant circadian clock:  how plants tell the time-perature.  Plant Signaling & Behavior 7, 1219-1223.
  24. Jones, M.A., Williams, B.A., McNicol, J.W., Simpson, C.G., Brown, J.W.S. & Harmer, S.L. 2012.  Mutation of Arabidopsis spliceosomal timekeeper locus 1 causes circadian clock defects.  The Plant Cell 24, 4066-4082.
  25. Kalyna, M., Simpson, C.G., Syed, N.H., Lewandowska, D., Marquez, Y., Kusenda, B., Marshall, J., Fuller, J., Cardle, L., McNicol, J.W., Dihn, H., Barta, A. & Brown, J.W.S. 2012.  Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis.  Nucleic Acids Research 40, 2454-2469.
  26. Marquez, Y., Brown, J.W.S., Simpson, C.G., Barta, A. & Kalyna, M. 2012.  Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis.  Genome Research 22, 1184-1195.
  27. Shaw, P.J. & Brown, J.W.S. 2012.  Nucleoli: composition, function and dynamics (Review).  Plant Physiology 158, 44-51.
  28. Streitner, C., Köster, T., Simpson, C.G., Shaw, P., Danisman, S., Brown, J.W.S. & Staiger, D. 2012.  An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana.  Nucleic Acids Research 40, 11240-11255.
  29. Syed, N.H., Kalyna, M., Marquez, Y., Barta, A. & Brown, J.W.S. 2012.  Alternative splicing in plants - coming of age.  Trends in Plant Science 17, 616-623.
  30. The International Barley Genome Sequencing Consortium, Waugh, R., Hedley, P.E., Liu, H., Morris, J., Russell, J., Druka, A., Marshall, D.F., Bayer, M. & Brown, J.W.S. 2012.  A physical, genetic and functional sequence assembly of the barley genome.  Nature 491, 711-716.
  31. Rakitina, D.V., Taliansky, M.E., Brown, J.W.S. & Kalinina, N.O. 2011.  Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates.  Nucleic Acids Research 39, 8869-8880.
  32. Kim, S.-H., Spensley, M., Choi, S.K., Calixto, C.P.G., Pendle, A.F., Koroleva, O., Shaw, P.J. & Brown, J.W.S. 2010.  Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli.  Nucleic Acids Research 38, 3054-3067.
  33. Raczynska, K.D., Simpson, C.G., Ciesiolka, A., Szewc, L., Lewandowska, D., McNicol, J.W., Szweykowska-Kulinska, Z., Brown, J.W.S. & Jarmolowski, A. 2010.  Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana.  Nucleic Acids Research 38, 265-278.
  34. Sanchez, S.E., Petrillo, E., Beckwith, E.J., Zhang, X., Rugnone, M.L., Hernando, C.E., Cuevas, J.C., Godoy Herz, M.A., Depetris-Chauvin, A., Simpson, C.G., Brown, J.W.S., Cerdán, P.D., Borevitz, J.O., Mas, P., Ceriani, M.F., Kornbilhtt, A.R. & Yanovsky, M.J. 2010.  A methyl transferase links the circadian clock to the regulation of alternative splicing.  Nature 468, 112-116.
  35. Simpson, C.G., Manthri, S., Raczynska, K.D., Kalyna, M., Lewandowska, D., Kusenda, B., Maronova, M., Szweykowska-Kulinska, Z., Jarmolowski, A., Barta, A. & Brown, J.W.S. 2010.  Regulation of plant gene expression by alternative splicing.  Biochemical Society Transactions 38, 667-671.
  36. Taliansky, M.E., Brown, J.W.S., Rajamäki, M.L., Valkonen, J.P.T. & Kalinina, N.O. 2010.  Involvement of the plant nucleolus in virus and viroid infections: Parallels with animal pathosystems.  Advances in Virus Research 77, 119-158.
  37. Cole, C., Sobala, A., Lu, C., Thatcher, S.R., Bowman, A., Brown, J.W.S., Green, P.J., Barton, G.J. & Hutvagner, G. 2009.  Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs.  RNA 15, 2147-2160.
  38. Kim, S.-H., Koroleva, O.A., Lewandowska, D., Pendle, A.F., Clark, G.P., Simpson, C.G., Shaw, P.J. & Brown, J.W.S. 2009.  Aberrant mRNA transcripts and the nonsense-mediated decay proteins UPF2 and UPF3 are enriched in the Arabidopsis nucleolus.  Plant Cell 21, 2045-2057.
  39. Koroleva, O.A., Calder, G., Pendle, A.F., Kim, S.-H., Lewandowska, D., Simpson, C.G., Jones, I.M., Brown, J.W.S. & Shaw, P.J. 2009.  Dynamic behaviour of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.  Plant Cell 21, 1592-1606.
  40. Koroleva, O.A., Brown, J.W.S. & Shaw, P.J. 2009.  Localization of eIF4A-III in the nucleolus and splicing speckles is an indicator of plant stress.  Plant Signaling & Behavior 4, 1148-1151.
  41. Brown, J.W.S. & Shaw, P.J. 2008.  The role of the plant nucleolus in pre-mRNA processing.  Current Topics in Microbiology and Immunology 326, 291-311.
  42. Brown, J.W.S., Marshall, D.F. & Echeverria, M. 2008.  Intronic noncoding RNAs and splicing.  Trends in Plant Science 13, 335-342.
  43. Brown, J.W.S. & Shaw, P.J. 2008.  The role of the plant nucleolus in pre-mRNA processing.  In: Reddy, A.S.N. & Golovkin, M. (eds.).  Nuclear Pre-mRNA Processing in Plants.  Springer, New York, Chapter 326, 291-311.
  44. Canetta, E., Kim, S.-H., Kalinina, N.O., Shaw, J., Adya, A.K., Gillespie, T., Brown, J.W.S. & Taliansky, M.E. 2008.  A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.  Journal of Molecular Biology 376, 932-937.
  45. Simpson, C.G., Lewandowska, D., Fuller, J., Maronova, M., Kalyna, M., Davidson, D., McNicol, J.W., Raczynska, D., Jarmolowski, A., Barta, A. & Brown, J.W.S. 2008.  Alternative splicing in plants.  Biochemical Society Transactions 36, 508-510.
  46. Simpson, C.G. & Brown, J.W.S. 2008.  U12-dependent intron splicing in plants.  Current Topics in Microbiology and Immunology 326, 61-82.
  47. Simpson, C.G., Fuller, J., Maronova, M., Kalyna, M., Davidson, D., McNicol, J.W., Barta, A. & Brown, J.W.S. 2008.  Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts.  The Plant Journal 53, 1035-1048.
  48. Simpson, C.G. & Brown, J.W.S. 2008.  U-12 dependent intron splicing in plants.  In: Reddy, A.S.N. & Golovkin, M. (eds.).  Nuclear Pre-mRNA Processing in Plants.  Springer, New York, Chapter 326, 61-82.
  49. Kim, S.-H., MacFarlane, S.A., Kalinina, N.O., Rakitina, D.V., Ryabov, E., Gillespie, T., Haupt, S., Brown, J.W.S. & Taliansky, M.E. 2007.  Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection.  Proceedings of the National Academy of Sciences, USA 104, 11115-11120.
  50. Kim, S.-H., Ryabov, E.V., Kalinina, N., Rakitina, D.V., Gillespie, T., MacFarlane, S.A., Haupt, S., Brown, J.W.S. & Taliansky, M.E. 2007.  Cajal bodies and the nucleolus are required for a plant virus systemic infection.  EMBO Journal 26, 2169-2179.