Organismal benefits of transcription speed control at gene boundaries

Abstract

RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto‐immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read‐through transcription. The accelerated transcription mutant NRPB2‐Y732F exhibits increased association with 5′ splice site (5′SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.

Dual‐color 3D‐dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei

“…Overall, histograms resemble those of the double-labeled RNAPIIS2p (Figure 4a,b), with 66% of all RNAPIIS2p/ROXY1 CBC values and 58% of the ROXY1/ RNAPIIS2p CBC values in the PC range (Figure 6a,b). Our data demonstrate a high degree of colocalization between ROXY1 and RNAPIIS2p, which is the prominent variant of the RNAPII CTD isoforms during transcription elongation and polyadenylation (Zhu et al, 2018;Kindgren et al, 2020;Leng et al, 2020). In summary, the CBC analyses reveal a strong colocalization of ROXY1 with RNAPIIS2p, a slightly weaker one with RNAPIIunp and a low one with RNA-PII5Sp.…”

“…However, recently, large-scale pNET-seq approaches with RNAPII isoform-specific antibodies increased our knowledge about distinct roles of the three different RNAPII variants in the plant transcription cycle. (Hajheidari et al, 2013;Hetzel et al, 2016;Zhu et al, 2018;Kindgren et al, 2020;Leng et al, 2020). We detected a high degree of colocalization between ROXY1 and RNAPIIunp, a variant that was shown to accumulate in promotor-proximal regions at transcription start sites (TSS) and decreases towards the 3 0 end (Zhu et al, 2018).…”

“…AC values of 33% and 22%, respectively, and PC values of 20% and 20%, respectively, for RNA-PIIS5p/ROXY1 and ROXY1/RNAPIIS5p indicate a mixed situation with correlation as well as AC between the two proteins. RNAPIIS5p is most abundant during co-transcriptional splicing (Zhu et al, 2018;Kindgren et al, 2020;Leng et al, 2020), suggesting a weak activity of ROXY1 in this process. Interestingly, of all investigated CBC projections, RNAPIIS2p and ROXY1 exhibit the highest degree of colocalization, supported by accumulation of red colocalization spots ( Figure 4g) and histograms with a strong peak at about 0.9 (Figure 4h).…”

“…As known from animals (Kindgren et al, 2020), there is increasing support that transcription and splicing are coupled in plants. RNAPIIS5p is the most prominent isoform at plant exon-intron boundaries and presumably regulating splicing activities (Zhu et al, 2018;Kindgren et al, 2020;Leng et al, 2020). The small ROXY1 proportion colocalizing with RNAPIIS5p proposes minor activities in co-transcriptional splicing processes.…”

First Come, First Served: Sui Generis Features of the First Intron

“…As described above, exonic and intronic DNA exhibit distinct nucleosome occupancy/chromatin structure, as well as DNA and histone depositions, all of which could define RNA polymerase II (Pol II) elongation speed. Due to high GC content and concentrated nucleosomes in the exonic region, elongation speed is slower than that in the intronic region ( Figure 1 , top) [ 38 , 39 ] As a result, AS is regulated by the speed of transcriptional machinery [ 11 , 40 , 41 , 42 , 43 ] A myriad of articles demonstrate that splicing in many cases takes place co-transcriptionally [ 39 , 44 , 45 , 46 ] and the speed of transcription elongation driven by Pol II affects splicing [ 40 , 41 , 47 ] In the case of plants, the rate of splicing appears to be much slower than that of yeast. Jia et al showed that by developing a nanopore-based method, more than half of the introns remain unspliced after Pol-II transcribed 1 kb past the 3′-splice site, and the authors called such introns post-transcriptionally spliced introns (pts introns) [ 48 ].…”