Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.     

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.     

Enhancements of energetic particles near the heliospheric termination shock

The spacecraft Voyager 1 is at a distance greater than 85 au from the Sun, in the vicinity of the termination shock that marks the abrupt slowing of the supersonic solar wind and the beginning of the extended and unexplored distant heliosphere. This shock is expected to accelerate 'anomalous cosmic rays', as well as to re-accelerate Galactic cosmic rays and low-energy particles from the inner Solar System. Here we report a significant increase in the numbers of energetic ions and electrons that persisted for seven months beginning in mid-2002. This increase differs from any previously observed in that there was a simultaneous increase in Galactic cosmic ray ions and electrons, anomalous cosmic rays and low-energy ions. The low-intensity level and spectral energy distribution of the anomalous cosmic rays, however, indicates that Voyager 1 still has not reached the termination shock. Rather, the observed increase is an expected precursor event. We argue that the radial anisotropy of the cosmic rays is expected to be small in the foreshock region, as is observed.     

A cryo-electron microscopic study of ribosome-bound termination factor RF2

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.     

Experimental validation of inter-subspecific genetic diversity in rice represented by the differences between the DNA sequences of ‘Nipponbare’ and ‘93-11’

The pedigrees of three sequenced rice cultivars were analyzed to show that a majority of the genetic composition of 'Nipponbare' originates from japonica cultivars while the minority originates from indica cultivars. In contrast, '93-11' is derived mainly from indica cultivars with a smaller contribution from japonica cultivars. All ancestors of 'Guang lu ai 4' appeared to be indica lines. A set of molecular markers (46 InDels and 53 SSRs) polymorphic between 'Nipponbare' and '93-11' were examined in 46 typical indica and 47 typical japonica cultivars selected from 443 accessions according to Cheng's index. All cultivars were divided into indica and japonica groups without overlapping when clustered by Cheng's index, InDels and SSRs. Much higher InDel and SSR diversity between groups than within groups implies that the marker polymorphisms between 'Nipponbare' and '93-11' represent a large proportion of inter-subspecific diversity. About 85% of indica cultivars and more than 90% of japonica cultivars were confirmed to have the same PCR banding patterns as '93-11' and 'Nipponbare', respectively. Some polymorphic loci between 'Nipponbare' and '93-11' cannot be validated in other indica and japonica cultivars, either as subspecies-specific but not predominant alleles, or alleles not specific between the two groups. It was concluded that molecular markers developed from sequence polymorphism between 'Nipponbare' and '93-11' often represent inter-subspecific diversity, although some exceptions were sensitive to either particular marker loci or particular cultivars.