Genetic and physical interactions involving the yeast nuclear cap-binding complex.

Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.     

Particulate phosphorus sedimentation at the river inflow to a lake

Sedimentation at the Krutynia River inflow to Lake Kujno was closely related to hydrological regime. The highest sedimentation rates, recorded in spring, decreased during summer by two orders of magnitude. Granulometric segregation of settling seston along the inflow zone was related to differentiation of phoshorus content. Smaller particles were richer in P, producing a gradient of increasing P concentrations in the settling material. A substantial loss of P from polyphosphate and various organic fractions was found after settlement of river suspensoids. Mechanisms of P losses are discussed and possibilities of P retention within the inflow zone are considered.     

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins

Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E^(28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.     

A complex pathway for 3' processing of the yeast U3 snoRNA

Mature U3 snoRNA in yeast is generated from the 3′-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3′ processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2–5p or Lsm8p, the normal 3′-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2–8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3′ processing. The Sm complex may function as a back-up to stabilise 3′ ends that are not protected by Lhp1p.     

Apoptotic signals induce specific degradation of ribosomal RNA in yeast

Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast.     

Can Chara control phosphorus cycling in Lake Łuknajno (Poland)?

Waters of Lake uknajno (623 ha, 3 m max. depth postglacial basin in Masurian Lakeland) are of meso-eutrophic type in spite of an intensive land impact and substantial phosphorus loading. The reason of this peculiarity is an abundance of 4 species of charophytes (Chara aculeolata, C. aspera, C. contraria and C. tomentosa) which dominate the submerged vegetation of the lake and yielded 417 g m^-2 dry biomass in summer 1991. The plants have high potential for phosphorus uptake and accumulation as was checked in laboratory experiments. Due to this potential Chara can outcompete planktonic algae in mixed cultures at different mineral P supplies.