The role of the putative 3′ end processing endonuclease Ysh1p in mRNA and snoRNA synthesis

Pre-mRNA 3′ end formation is tightly linked to upstream and downstream events of eukaryotic mRNA synthesis. The two-step reaction involves endonucleolytic cleavage of the primary transcript followed by poly(A) addition to the upstream cleavage product. To further characterize the putative 3′ end processing endonuclease Ysh1p/Brr5p, we isolated and analyzed a number of new temperature- and cold-sensitive mutant alleles. We show that Ysh1p plays a crucial role in 3′ end formation and in RNA polymerase II (RNAP II) transcription termination on mRNA genes. In addition, we observed a range of additional functional deficiencies in ysh1 mutant strains, which were partially allele-specific. Interestingly, snoRNA 3′ end formation and RNAP II termination were defective on specific snoRNAs in the cold-sensitive ysh1-12 strain. Moreover, we observed the accumulation of several mRNAs including the NRD1 transcript in this mutant. We provide evidence that NRD1 autoregulation is associated with endonucleolytic cleavage and that this process may involve Ysh1p. In addition, the ysh1-12 strain displayed defects in RNA splicing indicating that a functional link may exist between intron removal and 3′ end formation in yeast. These observations suggest that Ysh1p has multiple roles in RNA synthesis and processing.

The role of blood pressure in glaucoma

Although intraocular pressure (IOP) remains an important risk factor for glaucoma, it is clear that other factors can also influence disease development and progression. More recently, the role that blood pressure (BP) has in the genesis of glaucoma has attracted attention, as it represents a clinically modifiable risk factor and thus provides the potential for new treatment strategies beyond IOP reduction. The interplay between blood pressure and IOP determines the ocular perfusion pressure (OPP), which regulates blood flow to the optic nerve. If OPP is a more important determinant of ganglion cell injury than IOP, then hypotension should exacerbate the detrimental effects of IOP elevation, whereas hypertension should provide protection against IOP elevation. Epidemiological evidence provides some conflicting outcomes of the role of systemic hypertension in the development and progression of glaucoma. The most recent study showed that patients at both extremes of the blood pressure spectrum show an increased prevalence of glaucoma. Those with low blood pressure would have low OPP and thus reduced blood flow; however, that people with hypertension also show increased risk is more difficult to reconcile. This finding may reflect an inherent blood flow dysregulation secondary to chronic hypertension that would render retinal blood flow less able to resist changes in ocular perfusion pressure. Here we review both clinical and experimental studies that have attempted to clarify the relationships among blood pressure, OPP and blood flow autoregulation in the pathogenesis of glaucoma.

Chronic intraocular pressure elevation impairs autoregulatory capacity in streptozotocin-induced diabetic rat retina

Purpose: To assess ocular blood flow responses to acute IOP stress following 4 weeks of chronic IOP elevation in streptozotocin (STZ)-induced diabetic and control rats. We hypothesise that chronic IOP elevation for 4 weeks will further impair blood flow regulation in STZ-induced diabetic rats eyes. Methods: Two weeks following citrate buffer or STZ-injections chronic IOP elevation was induced in Long Evans rats via fortnightly intracameral injections of microspheres (15 μm) suspended in 5% polyethylene glycol. IOP was monitored daily. Electroretinography (ERG, -6.79-2.07 log cd s m-2) was undertaken at Week 4 to compare photoreceptor (RmPIII), ON-bipolar cell (Vmax) and ganglion cell dominant ERG [scotopic threshold response (STR)] components. 4 weeks post-chronic IOP induction, ocular blood flow (laser Doppler flowmetry) was measured in response to acute IOP challenge (10-100 mmHg, in 5 mmHg steps, each 3 min). Results: Four weeks of chronic IOP (mean ± S.E.M., citrate: 24.0 ± 0.3 to 30.7 ± 1.3 and STZ-diabetes: 24.2 ± 0.2 to 31.1 ± 1.2 mmHg) was associated with reduced photoreceptor amplitude in both groups (-25.3 ± 2.2% and -17.2 ± 3.0%, respectively). STZ-diabetic eyes showed reduced photoreceptor sensitivity (citrate: 0.5 ± 1.8%, STZ-diabetic: -8.1 ± 2.4%). Paradoxically ON-bipolar cell sensitivity was increased, particularly in citrate control eyes (citrate: 166.8 ± 25.9%, STZ-diabetic: 64.8 ± 18.7%). The ganglion cell dominant STR was not significantly reduced in STZ-diabetic rats. Using acute IOP elevation to probe autoregulation, we show that STZ-diabetes impaired autoregulation compared with citrate control animals. The combination of STZ-diabetes and chronic IOP elevation further impaired autoregulation. Conclusions: STZ-diabetes and chronic IOP elevation appear to be additive risk factors for impairment of ocular blood flow autoregulation.

Functional genomic analysis of systemic cell division regulation in legumes

Legumes develop root nodules from pluripotent stem cells in the rootpericycle in response to mitogenic activation by a decorated chitin-likenodulation factor synthesized in Rhizobium bacteria. The soybean genes encoding the receptor for such signals were cloned using map-based cloning approaches. Pluripotent cells in the root pericycle and the outer or inner cortex undergo repeated cell divisions to initiate a composite nodule primordium that develops to a functional nitrogen-fixing nodule. The process itself is autoregulated, leading to the characteristic nodulation of the upper root system. Autoregulation of nodulation (AON) in all legumes is controlled in part by a leucine-rich repeat receptor kinase gene (GmNARK). Mutations of GmNARK, and its other legume orthologues, result in abundant nodulation caused by the loss of a yet-undefined negative nodulation repressor system. AON receptor kinases are involved in perception of a long distance, root-derived signal, to negatively control nodule proliferation. GmNARK and LjHAR1 are expressed in phloem parenchyma. GmNARK kinase domain interacts with Kinase Associated Protein Phosphatase (KAPP). NARK gene expression did not mirror biological NARK activity in nodulation control, as q-RT-PCR in soybean revealed high NARK expression in roots, root tips, leaves, petioles, stems and hypocotyls, while shoot and root apical meristems were devoid of NARK RNA. High through-put transcript analysis in soybean leaf and root indicated that major genes involved in JA synthesis or response are preferentially down-regulated in leaf but not root of wild type, but not NARK mutants, suggesting that AON signaling may in part be controlled by events relating to hormone metabolism. Ethylene and abscisic acid insensitive mutants of L. japonicus are described. Nodulation in legumes has significance to global economies and ecologies, as the nitrogen input into the biosphere allows food, feed and biofuel production without the inherent costs associated with nitrogen fertilization [1]. Nodulation involves the production of a new organ capable of nitrogen fixation [2] and as such is an excellent system to study plant – microbe interaction, plant development, long distance signaling and functional genomics of stem cell proliferation [3, 4]. Concerted international effort over the last 20 years, using a combination of induced mutagenesis followed by gene discovery (forward genetics), and molecular/biochemical approaches revealed a complex developmental pathway that ‘loans’ genetic programs from various sources and orchestrates these into a novel contribution. We report our laboratory’s contribution to the present analysis in the field.