Abundance and distribution of RNA polymerase II in Arabidopsis interphase nuclei.

Veit Schubert,Klaus Weisshart

doi:10.1093/jxb/erv091

Abstract

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic protein-coding genes. Analysing the topological distribution and quantification of RNAPII can contribute to understanding its function in interphase nuclei. Previously it was shown that RNAPII molecules in plant nuclei form reticulate structures within euchromatin of differentiated Arabidopsis thaliana nuclei rather than being organized in distinct 'transcription factories' as observed in mammalian nuclei. Immunosignal intensity measurements based on specific antibody labelling in maximum intensity projections of image stacks acquired by structured illumination microscopy (SIM) suggested a relative proportional increase of RNAPII in endopolyploid plant nuclei. Here, photoactivated localization microscopy (PALM) was applied to determine the absolute number and distribution of active and inactive RNAPII molecules in differentiated A. thaliana nuclei. The proportional increase of RNAPII during endopolyploidization is confirmed, but it is also shown that PALM measurements are more reliable than those based on SIM in terms of quantification. The single molecule localization results show that, although RNAPII molecules are globally dispersed within plant euchromatin, they also aggregate within smaller distances as described for mammalian transcription factories.

Highlights

Most eukaryotic genes are transcribed by RNA polymerase II (RNAPII) (Kornberg, 1999; Sims et al, 2004)
By applying structured illumination microscopy (SIM), it has been proven that the relative amounts of RNAPII enzymes in differentiated 2C–32C leaf nuclei of A. thaliana proportionally increase with increasing endopolyploidy (Schubert, 2014)
To determine the number of RNAPII molecules per nucleus to a better extent and to check whether transcription factories may exist in plants, nuclei were labelled with specific antibodies against active and inactive RNAPII modifications and 3D-photoactivated localization microscopy (PALM) was applied in combination with SIM

Summary

Introduction

Most eukaryotic genes are transcribed by RNA polymerase II (RNAPII) (Kornberg, 1999; Sims et al, 2004). The quantity of active RNAPII in nuclei reflects the degree of transcription. Depending on its position on a gene and the stage of transcription, RNAPII is differentially phosphorylated. Its activation requires the phosphorylation at Ser and Ser of the heptapeptide YSPTSPS present as tandem repeats in the RNAPII CTD (Hirose and Ohkuma, 2007; Hajheidari et al, 2013). Antibodies specific for the phosphorylation state of the peptide allow the discrimination between active and inactive RNAPII (Bourdon et al, 2012). To initiate transcription and for the binding onto promoters of genes, phosphorylation at Ser is necessary (Hengartner et al, 1998; Komarnitsky et al, 2000). At the end of transcription, the Ser phosphorylation is removed, whereas RNAPIISer2ph accumulates at the 3ʹ end of the genes (Egloff and Murphy, 2008)

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