RNA polymerase II collision interrupts convergent transcription.

Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision.

The establishment of winter wheat root system architecture in field soils: The effect of soil type on root development in a temperate climate.

Winter wheat (Triticum aestivum L.) is an important cereal crop in the temperate climates of western Europe. Root system architecture is a significant contributor to resource capture and plant resilience. However, the impact of soil type on root system architecture (RSA) in field structured soils is yet to be fully assessed. This work studied the development of root growth using deep cultivation (250 mm) during the tillering phase stage (Zadock stage 25) of winter wheat across three soil types. The three sites of contrasting soil types covered a geographical area in the UK and Ireland in October 2018. Root samples were analysed using two methods: X-ray computed tomography (CT) which provides 3D images of the undisturbed roots in the soil, and a WinRHIZO™ scanner used to generate 2D images of washed roots and to measure further root parameters. Important negative relationships existed between soil bulk density and root properties (root length density, root volume, surface area and length) across the three sites. The results revealed that despite reduced root growth, the clay (Southoe) site had a significantly higher crop yield irrespective of root depth. The loamy sand (Harper Adams) site had significantly higher root volume, surface area and root length density compared with the other sites. However, a reduction in grain yield of 2.42 Mt ha-1 was incurred compared with the clay site and 1.6 Mt ha-1 compared with the clay loam site. The significantly higher rooting characteristics found in the loamy sand site were a result of the significantly lower soil bulk density compared with the other two sites. The loamy sand site had a lower soil bulk density, but no significant difference in macroporosity between sites (p > 0.05). This suggests that soil type and structure directly influence crop yield to greater extent than root parameters, but the interactions between both need simultaneous assessment in field sites.

Plasma phosphorylated-TDP-43 protein levels correlate with brain pathology in frontotemporal lobar degeneration.

In the present study, we have correlated plasma TDP-43 levels, as measured by ELISA, with the presence of TDP-43 pathological changes in the brains of 28 patients with frontotemporal lobar degeneration (FTLD) (14 with FTLD-TDP and 14 with FTLD-tau) and 24 patients with pathologically confirmed AD (8 with, and 16 without, TDP-43 pathological changes). Western blotting revealed full-length TDP-43, including a phosphorylated form, and a phosphorylated C-terminal fragment, in all samples examined. Both ELISA and immunohistochemistry were performed using phospho-dependent and phospho-independent TDP-43 antibodies for detection of phosphorylated and total TDP-43, respectively. Over all 52 cases, plasma levels of TDP-43, and scores of brain TDP-43 pathology, determined using TDP-43 phospho-dependent antibody correlated with the equivalent measure determined using the TDP phospho-independent antibody. In FTLD, but not AD, TDP-43 plasma levels correlated significantly with the pathology score when using the TDP-43 phospho-dependent antibody, but a similar correlation was not seen in either FTLD or AD using the TDP-43 phospho-independent antibody. With the TDP-43 phospho-independent antibody, there were no significant differences in median plasma TDP-43 levels between FTLD, or AD, patients with or without TDP-43 pathology. Using TDP-43 phospho-dependent antibody, median plasma TDP-43 levels were greater in patients with, than in those without, TDP-43 pathology for FTLD patients, though not significantly so, but not for AD patients. Present assays for TDP-43 do not differentiate between FTLD, or AD, patients with or without TDP-43 pathological changes in their brains. However, the levels of phosphorylated TDP-43 in plasma do correlate with the extent of TDP-43 brain pathology in FTLD, and therefore might be a useful surrogate marker for tracking changes in TDP-43 brain pathology during the course of this disease.

Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA.

Triptolide, a natural product extracted from the Chinese plant Tripterygium wilfordii, possesses antitumor properties. Despite numerous reports showing the proapoptotic capacity and the inhibition of NF-kappaB-mediated transcription by triptolide, the identity of its cellular target is still unknown. To clarify its mechanism of action, we further investigated the effect of triptolide on RNA synthesis in the human non-small cell lung cancer cell line A549. Triptolide inhibited both total RNA and mRNA de novo synthesis, with the primary action being on the latter pool. We used 44K human pan-genomic DNA microarrays and identified the genes primarily affected by a short treatment with triptolide. Among the modulated genes, up to 98% are down-regulated, encompassing a large array of oncogenes including transcription factors and cell cycle regulators. We next observed that triptolide induced a rapid depletion of RPB1, the RNA polymerase II main subunit that is considered a hallmark of a transcription elongation blockage. However, we also show that triptolide does not directly interact with the RNA polymerase II complex nor does it damage DNA. We thus conclude that triptolide is an original pharmacologic inhibitor of RNA polymerase activity, affecting indirectly the transcription machinery, leading to a rapid depletion of short-lived mRNA, including transcription factors, cell cycle regulators such as CDC25A, and the oncogenes MYC and Src. Overall, the data shed light on the effect of triptolide on transcription, along with its novel potential applications in cancers, including acute myeloid leukemia, which is in part driven by the aforementioned oncogenic factors.

Spontaneous pancreatic islet cell tumor in a black and white colobus monkey (Colobus guereza kikuyuensis).

A 12-year-old, male black and white colobus monkey (Colobus guereza kikuyuensis) from a small community zoo presented with a 6-month history of mild, slowly progressive ataxia and paresis culminating in an acute episode of recumbency, depression, and seizures. The animal was humanely euthanatized. Gross post-mortem examination revealed significant abnormalities including diffuse pallor of the carcass and a firm, pale, 8-cm diameter mass, adherent to the serosa of the proximal duodenum and colon, and embedded within the pancreas and mesenteric root. Histologically, the mass had characteristics of a neuroendocrine or endocrine tumor. Immunohistochemical stains for chromogranin, synaptophysin, insulin, and glucagon were positive, confirming the diagnosis of a mixed pancreatic islet cell tumor. These tumors are rare in all species except ferrets and unreported previously in colobus monkeys.