Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5′UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5′UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5′UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.     

Functional characterization of isolated RNA-binding domains of the GRSF1 protein

The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10 mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (K_D-value) of 0.5 μM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.     

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Old Man Saltbush mortality following fire challenges the resilience of post-mine rehabilitation in central Queensland,Australia

Landscape rehabilitation following mining is required to be resilient to disturbance impacts such as fire, drought and disease. As mining companies undergo the process of rehabilitation certification and mine closure, there are notable knowledge gaps on the ecological risks associated with mature rehabilitated landscapes, based largely on the assumption that rehabilitation is analogous to reference communities. However, the response to fire disturbance across a range of landscapes remains largely untested and in particular there is limited understanding of recovery traits of plant species that occur naturally or are commonly seeded into rehabilitation. In August 2018, a controlled fire was applied to 37 hectares of 12-year-old coal-mine rehabilitation in central Queensland, Australia. We used a combination of (i) ground plot surveys and (ii) drone imagery to compare the vegetation response of burnt woody species to unburnt controls prior to, and for, two years following the fire. The survival of the most dominant shrub species found on the rehabilitation site was significantly impacted by the fire. Old Man Saltbush (Atriplex nummularia Lindl. subsp. nummularia) recorded significant post-fire mortality, with ground surveys recording an average reduction of 89% of stems per hectare across the burnt site, while unburnt controls remained unchanged. The plot data analysis was supported with high spatial and temporal resolution drone imagery, classified using a Random Forest machine-learning approach. Change analysis of these maps showed a significant decline of 82% in Old Man Saltbush plant density and 92% reduction in foliage cover following the fire. In addition, the mean canopy area of individual Old Man Saltbush shrubs reduced significantly from a pre-fire mean of 11.3 to 4.8 m² two years following the fire. A spatial proximity analysis showed that those individuals that survived the fire were located significantly closer to unburnt areas and bare spoil, indicating that discontinuous ground fuel loads can greatly improve the survivability of individuals. This study provides new evidence on the contested fire sensitivity of Old Man Salt bush and demonstrates the risk that future climate-driven extreme events may have on the resilience of novel ecosystems.     

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Weitere Untersuchungen über die Befruchtungs- und Kreuzungsverhältnisse einiger Melilotusarten (Steinklee)

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